Combined nanoparticles, nucleic acid drug preparations, and preparation methods and applications thereof

Abstract

The present invention relates to a combination nanoparticle and its preparation method and application. The combination nanoparticle is prepared from polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer, cationic lipid, and ionizable lipid, wherein the mass proportion of the cationic lipid is greater than 0 but not more than 4%, the mass proportion of the ionizable lipid is 45% to 70%, and the mass proportion of the polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer is 26% to 55%. Combination nanoparticles are prepared from the combination nanoparticles and nucleic acid drugs. The combination nanoparticle preparation prepared by the present invention is used for carrying nucleic acid drugs, has high transport efficiency, and can stably express nucleic acids. It can be preferably used to prevent and treat tumors or viral infections.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to combined nanoparticles, nucleic acid drug preparations, and preparation methods and applications thereof. Background Art

[0002] Messenger RNA (mRNA) serves as the bridge between genes and proteins. It is a type of single-stranded RNA, formed by the polymerization of one strand of the double-stranded deoxyribonucleic acid (DNA) as a template and four ribonucleoside triphosphates (A, U, G, and C) under the catalysis of RNA polymerase, via phosphodiester bonds. In the cytoplasm, immature mRNA undergoes processing and modification through steps such as capping, tailing, and intron cleavage to become mature mRNA, which precisely directs protein synthesis in the cytoplasm. mRNA is translated intracellularly and does not enter the nucleus, eliminating the risk of integration into human DNA. Furthermore, it can target intracellular targets inaccessible to traditional small molecule drugs and antibody-based therapeutics. Therefore, using mRNA as a preventive and therapeutic agent offers significant advantages and potential for the prevention and treatment of a wide range of diseases. Currently, only two mRNA vaccines have been approved for use against the novel coronavirus, while mRNA drugs for other diseases, such as cancer, are still in clinical trials.

[0003] While mRNA nucleic acid drugs offer significant advantages and potential for regulating gene expression and preventing and treating malignant diseases, mRNA, as a single-stranded protein, is extremely unstable in vitro and under physiological conditions, susceptible to degradation by RNA nucleases (RNAases) in the air or blood. Furthermore, mRNA's negative charge prevents it from crossing cell membranes and entering the cell interior. Furthermore, mRNA has difficulty escaping endosomes and entering the cytoplasm to exert its effects. Furthermore, the uridine ribonucleoside (U) in mRNA is susceptible to immunogenicity, which in some cases may increase the potential toxic side effects of mRNA drugs. Specific delivery of mRNA to target cells requires addressing three key challenges: extracellular barriers, endosomal escape, and intracellular protein expression. Using delivery vehicles to deliver mRNA effectively overcomes these challenges. Currently, lipid nanoparticles (LNPs) are the most widely approved delivery system for clinical use. In recent years, research has deepened, resulting in significant breakthroughs in the selection and applicability of carrier materials. However, there are still many limitations in clinical application. For example, LNPs are mainly concentrated in the liver after administration, which may cause autoimmune hepatitis, and are difficult to expand to other parts of the body. Therefore, the development of new non-liver-targeted mRNA delivery vectors will become the key to the development of nucleic acid drugs.

[0004] In the research on constructing nucleic acid delivery vectors, the technical team of the present invention has continuously conducted research and breakthroughs from various aspects of the delivery platform to seek nucleic acid drugs that can be applied to clinical applications. The non-liver-targeted nucleic acid nanoparticles composed of polymers, cationic lipids, and ionizable lipids constructed in the early stage have achieved good non-liver-targeted tumor treatment effects. Moreover, the continuous preparation method based on microchannel reactors makes it possible for nucleic acid drugs to achieve convenient industrial production, which is a transformative and intelligent preparation technology for improving the production capacity of nucleic acid drugs. In further research, the technical team of the present invention found that the dosage of polymers, cationic lipids, and ionizable lipids has a great influence on the transport efficiency of mRNA in the body. In order to obtain better mRNA effects and improve the therapeutic effect on viruses, it is necessary to develop a nanoparticle preparation that can be stably expressed and has high transport efficiency. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a combined nanoparticle, a nucleic acid drug preparation using the combined nanoparticle as a carrier, and a preparation method thereof, wherein the nucleic acid in the drug preparation of the nanoparticle can be stably expressed and has high transport efficiency.

[0006] To achieve the above objectives, the following technical solutions are included.

[0007] The first aspect of the present invention is to provide a combined nanoparticle, which is prepared from polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer, cationic lipid, and ionizable lipid, wherein the mass proportion of the cationic lipid is greater than 0 but not more than 4%, the mass proportion of the ionizable lipid is 45% to 70%, and the mass proportion of the polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer is 26% to 55%.

[0008] In some of the embodiments, the polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer is polyethylene glycol-modified polylactide or polyethylene glycol-modified poly(glycolide-co-lactide), such as mPEG-PLGA polymer, wherein the molecular weight of PLGA is 2k-5k, more preferably 2k.

[0009] In some embodiments, the molecular weight of the polyethylene glycol is in the range of 1,000 to 10,000 Daltons.

[0010] In some of the embodiments, the mass proportion of the cationic lipid in the composite nanoparticles does not exceed 4%, preferably 0.5%-4%, preferably 0.5%-3%, or 0.5%-2%, 0.5%-1.5%.

[0011] In some of the embodiments, the mass proportion of the ionizable lipid in the composite nanoparticles is 50% to 70%, preferably 50% to 60%, or 50% to 58%, or 52% to 56%, or 54% to 56%.

[0012] In some of the embodiments, the mass proportion of the polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid-glycolic acid) copolymer in the combined nanoparticles is 26% to 50%, preferably 26% to 49%, more preferably 30% to 49%, or 35% to 46%, or further preferably 40% to 46%, or 43% to 45%.

[0013] In some embodiments, the cationic lipid material can be dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyldodecyl ammonium bromide (DTAB), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTMA), trimethylbromo(2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), trimethyltetradecyl ammonium bromide (TTAB), trimethylhexadecyl ammonium bromide (CTAB), dimethyldioctadecyl ammonium 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- One of hydroxybutyl-2,3-dioleyloxypropylammonium bromide (DORIE-HB), dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl-2,3-dicetadecyloxypropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2,3-dicetadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2,3-dicetadecyloxypropylammonium 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).

[0014] More preferably, the cationic lipid is one or more of DOTMA, DOTAP, DORI, DSRIE, DOGS, and DOSC, and most preferably is DOTAP.

[0015] In some embodiments, the ionizable lipid is Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, and may also be the following ionizable lipids:

[0016] BHEM-DBA: BHEM-APMP: BHEM-EAA: BHEM-AEA:

[0017] The second aspect of the present invention is to provide a combined nanoparticle preparation prepared using the above-mentioned combined nanoparticles as a carrier.

[0018] A combined nanoparticle preparation is prepared from the combined nanoparticles described above and a nucleic acid drug, wherein the mass ratio of the combined nanoparticles to the nucleic acid is 125-500:1.

[0019] In some of the embodiments, the mass ratio of the combined nanoparticles to the nucleic acid is preferably 200-400:1, or 220-300:1, or 230-290:1, or 230-280:1, or 240-270:1, or 240-260:1, more preferably 245-260:1.

[0020] In some embodiments, the nucleic acid is siRNA or mRNA, and in some embodiments, the siRNA or mRNA is a tumor treatment drug or an antiviral infection treatment drug, and more preferably, it is mRNA.

[0021] The third aspect of the present invention is to provide a method for preparing the above-mentioned combined nanoparticle preparation.

[0022] The preparation method of the combined nanoparticle preparation comprises the following steps:

[0023] S1. dissolving polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer, a cationic lipid, and an ionizable lipid in an organic solvent to obtain three solutions;

[0024] S2. The three solutions were added to ultrapure water, heated and stirred to prepare composite nanoparticles;

[0025] S3. Filtration, concentration, and ultrafiltration to obtain a suspension of combined nanoparticles from which the organic solvent has been removed;

[0026] S4. Mix the nucleic acid aqueous solution with the combined nanoparticle suspension of step S3, and incubate at room temperature to obtain a combined nanoformulation.

[0027] In some embodiments, the organic solvent is a solution of chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran, acetone, etc., preferably dimethyl sulfoxide or dimethylformamide.

[0028] In some of the embodiments, the heating temperature in step S2 is 55 degrees Celsius to 70 degrees Celsius, in some preferred embodiments, 60 degrees Celsius to 68 degrees Celsius, and more preferably, 62 degrees Celsius to 67 degrees Celsius.

[0029] In some embodiments, in step S4, the incubation temperature is 20 minutes to 40 minutes. The incubation time can be adjusted according to the conventional needs in the art and the preparation amount.

[0030] The fourth aspect of the present invention provides the use of the above-mentioned combined nanoparticle preparation in the preparation of nucleic acid drugs for preventing and treating diseases.

[0031] In some embodiments, the disease is caused by a viral infection.

[0032] In some of the embodiments, the nucleic acid drug is an mRNA vaccine.

[0033] In some of the embodiments, the nucleic acid of the combined nanoparticle formulation is mRNA expressing a novel coronavirus-specific antigen.

[0034] To prevent degradation of mRNA drugs and facilitate storage, the present invention differs from previous preparation methods in that, during preparation, polymers, cationic lipids, and ionizable lipids are first constructed into nanoparticles that serve as delivery vehicles. These are then simply mixed with the mRNA at the time of use, making them easy to use and store. During research, we discovered that the amounts of polymers, cationic lipids, and ionizable lipids used in nanoparticles prepared in this manner significantly impact the in vivo transport efficiency of mRNA. By comprehensively considering the various factors that influence the nanoparticles, and after relatively comprehensive considerations, we optimized the composition of each substance and achieved a combined nanoparticle formulation with high transport efficiency and stable nucleic acid expression for nucleic acid drugs, particularly mRNA drug delivery.

[0035] The present invention also developed a nanoparticle drug that uses nanoparticles to deliver the new coronavirus mRNA vaccine, which can induce the production of high levels of S protein-specific neutralizing antibodies in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the process flow of the combined nanoparticle preparation process of the present invention.

[0037] Figure 2The expression evaluation graph of firefly luciferase mRNA (Lui-mRNA) in vivo after intramuscular injection of the combined nanoparticles in Example 2; wherein, A is an in vivo expression evaluation graph of Lui-mRNA delivered by the combined nanoparticles prepared when the polymer is mPEG5K-PLGA5K, the mass proportion of DOTAP is 1% to 8%, and the mass proportion of Dlin-MC3-DMA is 10% to 70%, B is an in vivo expression evaluation graph of Lui-mRNA delivered by the combined nanoparticles prepared when the polymer is mPEG5K-PLGA12.5K, the mass proportion of DOTAP is 1% to 20%, and the mass proportion of Dlin-MC3-DMA is 10% to 70%, respectively. C, D, and E are in vivo expression evaluation graphs of Lui-mRNA delivered by the combined nanoparticles prepared when the polymer is mPEG2K-PLGA2K / mPEG2K-PLGA5K / mPEG2K-PLGA10K, the mass proportion of DOTAP is 1% to 4%, and the mass proportion of Dlin-MC3-DMA is 10% to 70%, respectively.

[0038] Figure 3 Graph showing the expression evaluation of firefly luciferase mRNA (Lui-mRNA) in vivo after intramuscular injection of the combined nanoparticles after component refinement in Example 4.

[0039] Figure 4 Figure 4 shows the evaluation of the expression of firefly luciferase mRNA (Lui-mRNA) in vivo after intramuscular injection of the combined nanoparticles incorporating different types of ionizable lipids.

[0040] Figure 5 Expression graphs of S protein-specific IgG antibody concentrations induced by the two combined nanoparticle delivery of mSRAS-CoV-2 in Example 5; wherein A is a standard curve image prepared for ELISA detection, and B is an expression graph of IgG antibody concentrations at different time points.

[0041] Figure 6 Expression diagram of neutralizing antibody production induced by combined nanoparticle delivery of mSRAS-CoV-2 in Example 6; wherein, A is a graph of specific IgG antibody titers after intramuscular injection at different doses, and B is a graph of pseudovirus neutralizing antibody levels after intramuscular injection at different doses. DETAILED DESCRIPTION

[0042] To facilitate 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 present disclosure more thorough and comprehensive.

[0043] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0045] In some preferred embodiments of the present invention, a composite nanoparticle is provided. When the nucleic acid carried is mRNA, the composite nanoparticle is prepared from polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer, cationic lipid, and ionizable lipid; wherein, in the composite nanoparticle, the mass proportion of the cationic lipid is 0.5%-4%, the mass proportion of the ionizable lipid is 45%-70%, and the mass proportion of the polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer is 26%-55%.

[0046] Furthermore, in the combined nanoparticles, the mass proportion of the cationic lipid has a greater impact on the performance of the entire nanoparticle and its preparation, preferably 0.5%-2%, the mass proportion of the ionized lipid is 45% to 70%, and the mass proportion of the polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic acid glycolic acid) copolymer is 38% to 49%.

[0047] In the combined nanoparticles, the mass proportion of the cationic lipid is 0.5%-1.5%, the mass proportion of the ionized lipid is 50%-60%, and the mass proportion of the polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid glycolic acid) copolymer is 38%-49%.

[0048] In some of the embodiments, the cationic lipid is DOTAP, the ionizable lipid is Dlin-MC3-DMA, and the polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer is PEG-PLGA.

[0049] When preparing the combined nanoparticles, the mass ratio of the combined nanoparticles to the nucleic acid is 220-300:1; in some embodiments, the mass ratio of the combined nanoparticles to the nucleic acid is 240-250:1.

[0050] The sources and processing methods of the raw materials used in the examples of the present invention are as follows.

[0051] The specific information of polyethylene glycol-modified aliphatic polyester is as follows: it was purchased from Jinan Daigang Biotechnology Co., Ltd. and purchased by Guangzhou Xinheng Huiheng;

[0052] Specific components Molecular weight (k) mpeg2k-plga50 / 50 2k 2k-2k mpeg2k-plga50 / 50 5k 2k-5k mpeg2k-plga50 / 50 10k 2k-10k

[0053] Organic solvents such as dimethyl sulfoxide (DMSO) and formaldehyde were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0054] Gelred nucleic acid gel dye: purchased from Biotium Company, and purchased by Guangzhou Cyclic Peptide Biotechnology Co., Ltd.

[0055] 6× loading dye: purchased from biotium company, and purchased by Guangzhou Cyclotide Biotechnology Co., Ltd.;

[0056] mSRAS-CoV-2 and Luciferase mRNA expressing SARS-CoV-2 S protein-specific antibodies were purchased from Hefei Afana Biotechnology Co., Ltd.

[0057] D-Luciferin potassium salt: Product No.: ST196, Manufacturer: Beyotime;

[0058] SARS-CoV-2 Omicron (B.1.1.529) variant Spike ELISA Kit, catalog number: KIT40591C, manufacturer: Sino Biological Company;

[0059] Dlin-MC3-DMA: Product number: 1224606-06-7, manufacturer: Aiweituo Biotechnology Co., Ltd.

[0060] 1,2-Dioleyloxypropyl)trimethylammonium chloride (DOTAP): Product No.: O02002, Manufacturer: Aiweituo Biotechnology Co., Ltd.;

[0061] Triton X-100, product number: 9002-93-1, manufacturer: Biofroxx, Germany;

[0062] Quant-iTTM RiboGreen@RNA Reagent and Kit: Manufacturer: New England Biolabs, USA;

[0063] Nanoparticle size and zeta potential analyzer, model: Nano ZSE, manufacturer: Malvern, UK;

[0064] Fully automatic small animal in vivo imaging device, manufacturer: PE Company, USA;

[0065] High-resolution field emission scanning electron microscope, model: Merlin, manufacturer: Carl Zeiss AG, Germany;

[0066] Heating magnetic stirrer, model: RCT basic, manufacturer: IKA, Germany;

[0067] Magnetic stirrer, model: C-MAG MS7, manufacturer: IKA, Germany;

[0068] Vertical electrophoresis tank: Model: VE-180, Manufacturer: Shanghai Tianneng Technology Co., Ltd.;

[0069] Electrophoresis apparatus: Model: EPS-300, Manufacturer: Shanghai Tianneng Technology Co., Ltd.

[0070] DMEM high glucose medium: Product number: C11995500BT, manufacturer: Gibco;

[0071] Fetal bovine serum: Product number: FSP500, manufacturer: Yikesai Biotechnology (Taicang) Co., Ltd.

[0072] Penicillin / streptomycin dual antibody solution: Product number: 15140-122, manufacturer: Gibco;

[0073] PBS phosphate buffer powder: Product number: ZLI9062, pH 7.2-7.4, manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0074] Multifunctional microplate reader, model: Varioskan LUX, manufacturer: Thermo Fisher Scientific (China) Co., Ltd.

[0075] Carbon dioxide cell culture incubator, model: Heracell VIOS 160i, manufacturer: Thermo Fisher Scientific (China) Co., Ltd. The present invention is further described in detail below with reference to specific examples.

[0076] Example 1. Process for preparing combined nanoparticles

[0077] like Figure 1The main components of the IKA magnetic stirrer shown include: a control panel (including touch buttons to adjust the speed, temperature, and time of the magnetic stirrer) and a heating plate (which can hold 10 to 15 round-bottom flasks and operate them simultaneously). The method for preparing nanoparticles by mixing with an IKA magnetic stirrer comprises the following steps: (1) taking out polyethylene glycol-poly(lactic-co-glycolic acid) copolymer mPEGnK-PLGAmK, cationic lipid DOTAP, and ionizable lipid from a -20°C refrigerator, and after returning to room temperature, using a balance to weigh a certain mass of each of them and dissolving them in analytical grade dimethyl sulfoxide (DMSO) to prepare material stock solutions of 20.0 mg / mL, 90.0 mg / mL, and 30.0 mg / mL, respectively; (2) placing a 25 mL round-bottom flask that has been treated with enzyme-free water in a 65°C oven for preheating, filling RNase-free ultrapure water in an enzyme-free 50 mL centrifuge tube, and placing the tube in a 65°C oven for preheating; (3) turning on the magnetic stirrer power supply, setting the speed to 900 rpm, first taking the volume of the organic phase (input by mass ratio) in 1.5 mL of water, and then adding the 1.5 mL of water; EP tube, and then use a pipette to add it once to a 25mL round-bottom flask preheated on a magnetic stirrer. After thorough mixing, use a 5mL pipette to add 5 times the volume of preheated RNAse-free ultrapure water at once, stir at 900rpm for about 1min, adjust the speed to 450rpm and continue stirring for about 10-15min; (4) transfer the particle suspension in the round-bottom flask to an ultrafiltration tube with a molecular weight cutoff of 100KDa (the new ultrafiltration tube is centrifuged with RNAse-free ultrapure water in advance to ensure that the water volume passes through the membrane completely), then fill the ultrafiltration tube with RNAse-free ultrapure water, balance it, place it in a centrifuge at 3500×g for 8min, discard the outer tube liquid, add RNAse-free ultrapure water to the ultrafiltration inner tube again and centrifuge it, repeat 3 times, and finally concentrate the combined nanoparticle suspension; (6) collect the combined nanoparticle suspension and use an enzyme-free pipette tip (100μL) to measure its concentrated volume (7) set the total mass of the organic phase material (W DOTAP+MC3+PEG-PLGA ) to mRNA mass ratio is 250:1, and mRNA is loaded according to the mass ratio of the mass of the organic phase material to the mass of mRNA of 250:1. That is, if the total mass of the prepared combined nanoparticle organic phase material is 2.5 mg, 10 μg of mRNA is added and incubated at room temperature for 30 minutes. Further, a combined nanoparticle preparation is obtained. The prepared mRNA-loaded nanosystem is named NP@ DOTAP / MC3 / PEG-PLGA mRNA.

[0078] Example 2, CLAN@ DOTAP / MC3 / PEG-PLGA Nanoparticle formulation optimization research

[0079] In order to explore and screen out the optimal nanoparticle formulation ratio, the present invention explored the effects of the feed ratio between lipid DOTAP, Dlin-MC3-DMA and copolymer and the molecular weight of the copolymer on the size, dispersity, potential and in vivo protein expression of the combined nanoparticles. This experiment used the total mass of the fixed organic phase material (W DOTAP+MC3+PEG-PLGA ) to mRNA mass ratio was 250:1, the total mass of the organic phase material of the nanoparticles was set to 2.5 mg, and the total mass of DOTAP, Dlin-MC3-DMA and PEGnK-PLGAmK accounted for 100%.

[0080] First, the copolymer mPEG5K-PLGA12.5K (mPEG block molecular weight of 5000, PLGA block molecular weight of 125000) was selected. The DOTAP and Dlin-MC3-DMA dosages were varied to investigate the effect of the DOTAP to Dlin-MC3-DMA ratio on the nanoparticles. As shown in Table 1, the DOTAP ratio significantly affected the potential of the nanoparticles. When the DOTAP mass ratio was 1%, the potential of the nanoparticles was negative. At lower DOTAP mass ratios, the particle dispersion decreased. When the DOTAP mass ratio was above 4%, the potential of the nanoparticles was positive. In particular, when the DOTAP mass ratio was above 8%, the potential of the nanoparticles was generally above 8mV. The potential increased with the higher Dlin-MC3-DMA mass ratio. The prepared nanoparticles had a particle size range of 110 to 160nm.

[0081] Table 1. The copolymer is mPEG5K-PLGA12.5K. The effect of changing the feeding amount of DOTAP and Dlin-MC3-DMA on the particle size (Diameter), distribution (PDI) and potential (Zeta potential) of the prepared nucleic acid nanocarriers.

[0082]

[0083]

[0084] Based on these results, the copolymer mPEG5K-PLGA5K was selected, and the DOTAP (refined to a DOTAP mass fraction of 8% or less) and Dlin-MC3-DMA dosage were varied to investigate the effect of the DOTAP to Dlin-MC3-DMA ratio on nanoparticle properties. As shown in Table 2, the mPEG5K-PLGA5K copolymer significantly impacted properties such as particle size. When the DOTAP mass fraction did not exceed 4%, the nanoparticle potential was almost negative. The resulting nanoparticles ranged in size from 60 to 110 nm.

[0085] Table 2. The copolymer is mPEG5K-PLGA5K, the effect of changing the feeding amount of Dlin-MC3-DMA and refining the mass proportion of DOTAP on the particle size (Diameter), distribution (PDI) and potential (Zeta potential) of the prepared nucleic acid nanocarriers.

[0086]

[0087]

[0088] Based on the above-refined DOTAP mass fraction results, we continued to select a DOTAP mass fraction of 4% or less and varied the copolymer molecular weight to investigate its effect on nanoparticle properties. As shown in Tables 3, 4, and 5, the copolymer molecular weight influences the nanoparticle properties. As shown in Table 3, when the PLGA block molecular weight in the mPEG-PLGA polymer is 2K and the Dlin-MC3-DMA mass fraction is the same, the nanoparticles with a higher DOTAP mass fraction have larger particle sizes. At the same DOTAP mass fraction, the higher the Dlin-MC3-DMA mass fraction, the larger the nanoparticle size. As shown in Table 4, when the PLGA block molecular weight in the mPEG-PLGA polymer is 5K and the DOTAP mass fraction is the same, the Dlin-MC3-DMA mass fraction has no effect on the nanoparticle size. However, at the same Dlin-MC3-DMA mass fraction, the nanoparticles with a higher DOTAP mass fraction have larger particle sizes. As shown in Table 5, the mass proportion of DOTAP and the mass proportion of Dlin-MC3-DMA have no effect on the particle size of the nanoparticles; however, compared with the nucleic acid nanocarriers prepared with PLGA block molecular weights of 2K and 5K in the mPEG-PLGA polymer, the nanoparticle size is relatively large.

[0089] Table 3. Effect of the molecular weight of the PLGA block in the mPEG-PLGA polymer at 2K (the molecular weight of the PEG block was 2K and remained unchanged) on the particle size (Diameter), distribution (PDI) and zeta potential (Zeta potential) of the prepared nucleic acid nanocarriers.

[0090]

[0091]

[0092] Table 4. Effect of the molecular weight of the PLGA block of 5K in the mPEG-PLGA polymer (the molecular weight of the PEG block is 2K and remains unchanged) on the particle size (Diameter), distribution (PDI) and zeta potential of the prepared nucleic acid nanocarriers.

[0093]

[0094] Table 5. Effect of PLGA block molecular weight of 10K (PEG block molecular weight of 2K, unchanged) in mPEG-PLGA polymer on particle size (Diameter), distribution (PDI) and zeta potential (Zeta potential) of prepared nucleic acid nanocarriers.

[0095]

[0096]

[0097] Example 3: Verification of in vivo transfection efficiency of mRNA-loaded combined nanoparticles

[0098] In order to screen the component ratio of the combined nanoparticles of the present invention with the highest mRNA transport efficiency in vivo, this experiment used luciferase-expressing mRNA (Luci-mRNA) as a model to investigate the mRNA delivery and expression efficiency of intramuscular injection. DOTAP / MC3 / PEG-PLGALuci-mRNA; According to the formula of Example 2, a total of 133 species were prepared, and the preparation method was similar to that of Example 1: (1) Polyethylene glycol-poly (lactic acid glycolic acid) copolymer, i.e., mPEGnK-PLGAmK (mPEG block molecular weight is 5000 / 2000, PLA block molecular weight is 125000 / 10000 / 5000 / 2000), cationic lipid DOTAP, and ionizable lipid Dlin-MC3-DMA were taken out from -20°C, and after they returned to room temperature, 100.0 mg, 450.0 mg, and 150.0 mg were weighed, respectively, and dissolved in analytical grade dimethyl sulfoxide to prepare 20.0 mg / mL, 90.0 mg / mL, and 30.0 mg / mL, respectively. mg / mL material stock solution; (2) Dispense RNAse-free ultrapure water into enzyme-free 50mL centrifuge tubes and preheat in an oven at 65 degrees Celsius; (3) Turn on the power of the magnetic stirrer and set the speed to 900rpm. According to the mass ratios in Table 1, Table 2, Table 3, Table 4, and Table 5, convert them into actual feed volume as shown in Table 6 (prepared by 5 times volume expansion), first take the corresponding volume of organic phase into a 1.5mL EP tube, and then add it to a 25mL round-bottom flask placed on a magnetic stirrer at one time. After thorough mixing, use a 5mL pipette to add 5 times the volume of preheated RNAse-free ultrapure water, i.e. 2923.2μL, at one time. After stirring at 900rpm for about 1min, adjust the speed to 450rpm and continue stirring for about 10-15min; (4) Transfer the particle suspension in the round-bottom flask to an ultrafiltration tube with a molecular weight cutoff of 100KDa ((The new ultrafiltration tube was pre-treated with RNAse-free ultrapure water) A enzyme ultrapure water was centrifuged to allow the water to completely pass through the membrane), and then filled with RNAse-free ultrapure water in the ultrafiltration tube. After balancing, it was placed in a centrifuge at 3500×g for 8 minutes, and the liquid in the outer tube was discarded. RNAse-free ultrapure water was added to the ultrafiltration inner tube again and centrifuged. This was repeated 3 times, and the combined nanoparticle suspension was concentrated for the last time; (6) Different combined nanoparticle suspensions were collected and their concentrated volumes were measured using an enzyme-free pipette tip (100 μL). (7) The total mass of the organic phase material (W) was set. DOTAP+MC3+PEG-PLGA ) to mRNA mass ratio of 250:1, according to the mass ratio of the organic phase material to the mRNA mass ratio of 250:1 for loading mRNA. That is, the concentrated volume of NP with different ratios prepared by 5 times the volume is 444.4μL, then 373.3μL of particles are taken, 42μg Luci-mRNA is added, and the nanoformulation NP@ is obtained by incubation at room temperature for 30min. DOTAP / MC3 / PEG-PLGA Luci-mRNA.

[0099] Table 6. Volume of feedstock for the preparation of mPEG2K-PLGA2K, DOTAP, and Dlin-MC3-DMA

[0100]

[0101]

[0102] Using Luci-mRNA as a model, the transfection efficiency of the above-mentioned mRNA-loaded nanoparticles with different ratios in mice was investigated. DOTAP / MC3 / PEG-PLGA After Luci-mRNA was injected intramuscularly into the immunized animals, the mice were subjected to in vivo imaging at 12 h, and NP@ DOTAP / MC3 / PEG-PLGA The expression efficiency of Luci-mRNA after immunization. The experiment set up a PBS group and a drug-administered group, with 2 mice in each group, and each mouse was injected with 10μg of mLuc-NPs in the left and right legs; 12 hours after the mice were immunized, the mice were anesthetized by intraperitoneal injection of 125μL of sodium pentobarbital (1%); followed by injection of 200μL of luciferase substrate (15mg / mL) through the tail vein, and the luciferase fluorescence signal was collected for 60s using an IVIS spectrometer (PerkinElmer), and the fluorescence signal in the organ region (ROI) was quantified using Living Image 3.0. The experiment showed that: First, through intramuscular administration, the various ratios of the combined nanoparticles mLuc-NPs prepared by the above method can successfully deliver mRNA into the body and achieve the expression of the target protein ( Figure 2 ); second, the expression of the target protein in the nanoformulation prepared by the mPEG-PLGA polymer with a PLGA block molecular weight of 5K (the PEG block molecular weight was 5K and remained unchanged) was higher than that in the nanoformulation prepared by the mPEG-PLGA polymer with a PLGA block molecular weight of 12.5K (the PEG block molecular weight was 5K and remained unchanged), and when the mass proportion of DOTAP did not exceed 4%, the expression level of the nanoformulation in vivo was higher ( Figure 2 AB); Third, the expression of target protein in the nanoformulation prepared with a PLGA block molecular weight of 2K in the mPEG-PLGA polymer (the PEG block molecular weight was 2K and remained unchanged) was higher than that of the nanoformulation prepared with a PLGA block molecular weight of 5K and 10K in the mPEG-PLGA polymer (the PEG block molecular weight was 2K and remained unchanged) ( Figure 2 CDE); Fourth, in the nanoformulations prepared using mPEG2K-PLGA2K polymer, the lower the DOTAP mass ratio (i.e., 1%), the protein expression level was significantly higher when the Dlin-MC3-DMA mass ratio was 50% to 70% than that of the other nanoformulations ( Figure 2 C).

[0103] Example 4, preferably CLAN@ DOTAP / MC3 / PEG-PLGA Further refinement of nanoparticle formulations

[0104] In order to further optimize the optimal component ratio of the combined nanoparticles of the present invention for the highest in vivo mRNA transport efficiency, the copolymer mPEG2K-PLGA2K polymer was selected to prepare nanoparticles based on the above results, wherein the mass proportion of DOTAP was set to 0.5%, 0.75%, and 1%, and the mass proportion of Dlin-MC3-DMA was set to 45%, 50%, 55%, 60%, and 65%. The preparation method was similar to that in Example 1: (1) Polyethylene glycol-poly (lactic acid glycolic acid) copolymer, i.e., mPEGnK-PLGAmK (mPEG block molecular weight of 2000, PLA block molecular weight of 2000), cationic lipid DOTAP, and ionizable lipid Dlin-MC3-DMA were taken out from -20°C, and after they returned to room temperature, 100.0 mg, 450.0 mg, and 150.0 mg were weighed and dissolved in analytical grade dimethyl sulfoxide to prepare 20.0 mg / mL, 90.0 mg / mL, and 30.0 mg / mL, respectively. mg / mL material stock solution; (2) Dispense RNAse-free ultrapure water into enzyme-free 50mL centrifuge tubes and preheat in an oven at 65 degrees Celsius; (3) Turn on the power of the magnetic stirrer, set the speed to 900rpm, prepare according to the mass ratio in Table 7, and the actual feed volume is as shown in Table 8 (prepared by 5 times the volume). First, take the corresponding volume of organic phase into a 1.5mL EP tube, and then add it to a 25mL round-bottom flask placed on a magnetic stirrer at one time. After fully mixing, use a 5mL pipette to add 5 times the volume of preheated RNAse-free ultrapure water, i.e. 2923.2μL, at one time. After stirring at 900rpm for about 1min, adjust the speed to 450rpm and continue stirring for about 10-15min; (4) Transfer the particle suspension in the round-bottom flask to an ultrafiltration tube with a molecular weight cutoff of 100KDa ((The new ultrafiltration tube was pre-treated with RNAse-free ultrapure water in advance) A enzyme ultrapure water was centrifuged to allow the water to completely pass through the membrane), and then filled with RNAse-free ultrapure water in the ultrafiltration tube. After balancing, it was placed in a centrifuge at 3500×g for 8 minutes, and the liquid in the outer tube was discarded. RNAse-free ultrapure water was added to the ultrafiltration inner tube again and centrifuged. This was repeated 3 times, and the combined nanoparticle suspension was concentrated for the last time; (6) Different combined nanoparticle suspensions were collected and their concentrated volumes were measured using an enzyme-free pipette tip (100 μL). (7) The total mass of the organic phase material (W) was set. DOTAP+MC3+PEG-PLGA ) to mRNA mass ratio of 250:1, according to the mass ratio of the organic phase material to the mRNA mass ratio of 250:1 for loading mRNA. That is, the concentrated volume of NP with different ratios prepared by 5 times the volume is 444.4μL, then 373.3μL of particles are taken, 42μg Luci-mRNA is added, and the nanoformulation NP@ is obtained by incubation at room temperature for 30min. DOTAP / MC3 / PEG-PLGA Luci-mRNA.

[0105] Table 7. Particle size (Diameter), distribution (PDI), and zeta potential (Zeta potential) of nucleic acid nanocarriers prepared by using mPEG2K-PLGA2K and refining the mass ratio of DOTAP and Dlin-MC3-DMA.

[0106]

[0107]

[0108] Table 8. The feed volume when mPEG2K-PLGA2K was selected and the mass ratio of DOTAP and Dlin-MC3-DMA was refined.

[0109]

[0110] Using Luci-mRNA as a model, the transfection efficiency of the above mRNA-loaded nanoparticles in mice was investigated according to the administration method of Example 3. DOTAP / MC3 / PEG-PLGA After Luci-mRNA was injected intramuscularly into the immunized animals, the mice were subjected to in vivo imaging at 12 h, and NP@ DOTAP / MC3 / PEG-PLGA The expression efficiency of Luci-mRNA after immunization. The experiment set up a PBS group and a drug-administered group, with 2 mice in each group, and each mouse was injected with 10μg of mLuc-NPs in the left and right legs; 12h after the mice were immunized, the mice were anesthetized by intraperitoneal injection of 125μL sodium pentobarbital (1%); then 200μL of luciferase substrate (15mg / mL) was injected through the tail vein, and the luciferase fluorescence signal was collected for 60s using an IVIS spectrometer (PerkinElmer), and the fluorescence signal in the organ region (ROI) was quantified and analyzed using Living Image 3.0. The experiment showed that when the mPEG2K-PLGA2K polymer was used to prepare nanoformulations, the protein expression level was significantly higher when the mass proportion of DOTAP was 1% and the mass proportion of Dlin-MC3-DMA was 55% than that of the other nanoformulations ( Figure 3 ).

[0111] At the same time, in order to further verify that the combined nanoparticles with the optimal component ratio have high mRNA transport efficiency in vivo, according to the above results, 44% by mass of mPEG2K-PLGA2K and 1% DOTAP were selected, and the other two ionizable lipids SM-102 and ACL-0315 were used at a mass ratio of 55% to prepare nanoparticles. The preparation method was similar to that in Example 4: (1) Polyethylene glycol-poly (lactic acid glycolic acid) copolymer, i.e., mPEGnK-PLGAmK (mPEG block molecular weight of 2000, PLA block molecular weight of 2000), cationic lipid DOTAP, ionizable lipid SM-102 or ACL-0315 were taken out from -20°C, and after they returned to room temperature, 100.0 mg, 450.0 mg, and 150.0 mg were weighed and dissolved in analytical grade dimethyl sulfoxide to prepare 20.0 mg / mL, 90.0 mg / mL, and 30.0 mg / mL, respectively. mg / mL material stock solution; (2) Dispense RNAse-free ultrapure water into enzyme-free 50mL centrifuge tubes and preheat in an oven at 65 degrees Celsius; (3) Turn on the power of the magnetic stirrer and set the speed to 900rpm. Prepare according to the mass ratio in Table 7. The actual feed volume is as shown in Table 8 (prepared by 5 times the volume). First, take the corresponding volume of organic phase into a 1.5mL EP tube, and then add it to a 25mL round-bottom flask placed on a magnetic stirrer. After thorough mixing, use a 5mL pipette to add 5 times the volume of preheated RNAse-free ultrapure water, i.e. 2923.2μL, at one time. After stirring at 900rpm for about 1min, adjust the speed to 450rpm and continue stirring for about 10-15min; (4) Transfer the particle suspension in the round-bottom flask to an ultrafiltration tube with a molecular weight cutoff of 100KDa ((The new ultrafiltration tube was pre-treated with RNAse-free ultrapure water). A enzyme ultrapure water was centrifuged to allow the water to completely pass through the membrane), and then filled with RNAse-free ultrapure water in the ultrafiltration tube. After balancing, it was placed in a centrifuge at 3500×g for 8 minutes, and the liquid in the outer tube was discarded. RNAse-free ultrapure water was added to the ultrafiltration inner tube again and centrifuged. This was repeated 3 times, and the combined nanoparticle suspension was concentrated for the last time; (6) Different combined nanoparticle suspensions were collected and their concentrated volumes were measured using an enzyme-free pipette tip (100 μL). (7) The total mass of the organic phase material (W) was set. DOTAP+SM-102 / ACL-0315+PEG-PLGA ) to mRNA mass ratio of 250:1, according to the mass ratio of the organic phase material to the mRNA mass ratio of 250:1 for loading mRNA. That is, the concentrated volume of NP with different ratios prepared by 5 times the volume is 444.4μL, then 373.3μL of particles are taken, 42μg Luci-mRNA is added, and the nanoformulation NP@ is obtained by incubation at room temperature for 30min. DOTAP / SM-102\ACL-0315 / PEG-PLGA Luci-mRNA.

[0112] Using Luci-mRNA as a model, the transfection efficiency of the combined nanoparticles with the optimal composition ratio (w / w / w DOTAP:SM-102 / ACL-0315:PEG2K-PLGA2K=1:55:44) under different ionizable lipids was investigated according to the administration method of Example 3. DOTAP / SM-102\ACL-0315 / PEG-PLGA After Luci-mRNA was injected intramuscularly into the immunized animals, the mice were subjected to in vivo imaging at 12 h, and NP@ DOTAP / SM-102\ACL-0315 / PEG-PLGA Luci-mRNA expression efficiency after immunization. The experiment set up a PBS group and a drug-administered group, with 2 mice in each group, and each mouse was injected with 10μg of mLuc-NPs in the left and right legs; 12h after the mice were immunized, the mice were anesthetized by intraperitoneal injection of 125μL of sodium pentobarbital (1%); then 200μL of luciferase substrate (15mg / mL) was injected through the tail vein, and the luciferase fluorescence signal was collected for 60s using an IVIS spectrometer (PerkinElmer), and the fluorescence signal in the organ region (ROI) was quantified and analyzed using Living Image 3.0. The experiment showed that the optimal mass ratio of the combined nanoparticles, DOTAP: ionizable lipids: PEG2K-PLGA2K = 1:55:44, was applicable to the other ionizable lipids, and the prepared nanoformulations were able to successfully deliver mRNA into the body and achieve efficient expression of the target protein ( Figure 4 ).

[0113] Example 5: Different combinations of nanoparticles delivering mSRAS-CoV-2 induce the production of S protein-specific antibodies

[0114] In order to investigate the differences in the effects of different combinations of nanoparticles of the present invention in the prevention and treatment of the new coronavirus, this experiment used female healthy BALB / c mice as a model and first investigated the differences in the expression of specific antibodies in the body after intramuscular injection of different combinations of nanovaccines.

[0115] Two nanoparticle vaccines CLAN@ prepared from PEG-PLGA, cationic DOTAP, and ionizable lipid Dlin-MC3-DMA were listed. DOTAP / MC3 / PEG-PLGA 1-55-49 with CLAN@ DOTAP / MC3 / PEG-PLGA 4-30-66(The subscript numbers indicate the mass proportion of each component, wherein the molecular weight of the mPEG block is 2000 and the molecular weight of the PLGA block is 2000). The method for preparing the nano vaccine is as shown in Example 1, specifically: As described in Example 1, the steps for preparing the nano vaccine include: first taking the corresponding organic phase (412.5 μL mPEG2K-PLGA2K, 5.6 μL DOTAP, 41.7 μL DMSO and 125 μL Dlin-MC3-DMA) in a volume of 1.5 mL according to the feeding amounts in Tables 6 and 7 (5-fold volume amplification preparation); EP tube, and then added to a 25mL round-bottom flask placed on a magnetic stirrer. After thorough mixing, 5 times the volume of preheated RNAse-free ultrapure water, i.e., 2923.2μL, was added at one time using a 5mL pipette. After stirring at 900rpm for about 1min, the speed was adjusted to 450rpm and stirring was continued for about 10-15min; wherein the concentration of the polymer PEG2K-PLGA2K was 20mg / mL, the concentration of the cationic lipid DOTAP was 90mg / mL, and the concentration of the ionizable lipid Dlin-MC3-DMA was 30mg / mL; the nanovaccine CLAN@ was prepared according to the method in Example 4. DOTAP / MC3 / PEG-PLGA 4-30-66 with CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49 8240504 375012494

[0116] CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49 mRNA and CLAN@ DOTAP / MC3 / PEG-PLGA 4-30-66 Female BALB / c mice were immunized intramuscularly with the mRNA two-group nanovaccine group (2 μg / mouse) and 1×PBS buffer, and boosted immunization was performed on day 21. Orbital blood was collected from mice using capillaries 14, 28, and 42 days after administration. Orbital blood samples were centrifuged twice at 12,000 rpm for 10 minutes at 4°C. The serum was collected and stored at -80°C for further testing of the specific IgG antibody level produced in the serum using ELISA. Six female BALB / c mice were included in each group.

[0117] Experimental conclusion: Through intramuscular injection of CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49 mRNA or CLAN@ DOTAP / MC3 / PEG-PLGA 4-30-66 mRNA can induce the expression of S protein-specific antibodies at low doses, especially the optimal ratio component CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49 mRNA showed to induce higher levels of IgG antibodies ( Figure 5 ).

[0118] Example 6: Different doses of combined nanoparticles delivering mSRAS-CoV-2 induce neutralizing antibody production

[0119] In order to investigate the IgG antibody dose effect and neutralizing antibody level of the combined nanovaccine of the present invention in the prevention and treatment of novel coronavirus, this study used female healthy BALB / c mice as a model and investigated the effects of intramuscular injection of different doses of CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49 Differences in neutralizing antibody levels in vivo after mRNA combination nanovaccines. DOTAP / MC3 / PEG-PLGA 1-55-49 The mRNA preparation method is as described in Example 1: the preparation steps include: according to the feeding amount table 7 (5-fold volume amplification preparation), first take the corresponding organic phase (337.5 μL mPEG2K-PLGA2K, 1.4 μL DOTAP, 58.3 μL DMSO and 187.5 μL Dlin-MC3-DMA) volume into a 1.5 mL EP tube, and then add it once to a 25 mL round-bottom flask placed on a magnetic stirrer. After thorough mixing, use a 5 mL pipette to add 5 times the volume of preheated RNAse-free ultrapure water, i.e., 2923.2 μL, at one time. After stirring at 900 rpm for about 1 minute, adjust the speed to 450 rpm and continue stirring for about 10-15 minutes; wherein the concentration of polymer PEG2K-PLGA2K is 20 mg / mL, the concentration of cationic lipid DOTAP is 90 mg / mL, and the concentration of ionizable lipid Dlin-MC3-DMA is 30 mg / mL; prepared according to the method in Example 1, the nano vaccine CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49 , respectively, by intramuscular immunization of 10μg / mouse and 30μg / mouse CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49mRNAThe combined nano-vaccine and 1×PBS buffer were administered to female BALB / c mice, and booster immunization was performed on day 21. 42 days after administration, orbital blood was collected from the mice using capillaries. The orbital blood samples were centrifuged twice at 12,000 rpm for 10 minutes at 4°C, and the serum was collected and stored at -80°C. The concentration of specific IgG antibodies produced in the serum was further tested using ELISA. At the same time, the neutralizing antibodies in the serum samples on day 42 were determined by a pseudovirus neutralization experiment based on vesicular stomatitis virus (VSV), specifically: (1) The inactivated serum sample was taken out of the -80°C refrigerator and thawed on ice for later use. If it was not inactivated, it was inactivated at 56°C for 30 minutes. (2) Dilution: Dilute the serum sample with DMEM medium (without fetal bovine serum) and dilute it into 6 concentration gradients in a transparent 96-well plate: 1:10, 1:30, 1:90, 1:270, 1:810, 1:2430. For 1:10 dilution: add 32 μl to 288 μl of culture medium, with a total volume of 320 μl, and pipette 5 times to mix. For other dilutions: add 90 μl of the 1:10 diluted serum to 180 μl of culture medium, pipette 5 times, and dilute in sequence. Transfer the diluted serum to a white 96-well plate. Add 100 μl of diluted serum sample to each well of the white 96-well plate, and repeat 2 wells. (3) Blank control group CC: add 100 μl of DMEM medium, positive control group VC: add 100 μl of DMEM medium (4) Prepare DMEM complete medium (FBS + double antibody) (5) Thaw pseudovirus: Take pseudovirus out of -80℃ freezer and thaw quickly in room temperature water bath, dilute pseudovirus to 1.3×104TCID50 / ml with DMEM complete medium, add 50 μl of diluted virus to the experimental well and VC well, add 50 μl of DMEM complete medium to CC well. (6) Place the 96-well plate (without shaking to mix) in a 37℃, 5% CO2 incubator and incubate for 1 hour. (7) Digest Vero cells during incubation and adjust the cell density to 0.5×106cells / ml. After incubation, add 100 μl of cells to each well (cell number is 5×104cells / well) and place in a 37℃, 5% CO2 incubator to incubate for 24 hours. (8) After 24 h, remove the 96-well plate and equilibrate it to room temperature. Aspirate 150 μl of supernatant from the plate and add 100 μl of Bio-Lite luciferase detection reagent equilibrated at room temperature. Incubate for 3 min and immediately measure the luminescence value (RLU) using a microplate reader (chemiluminescence, do not shake).(9) Inhibition rate = (1-(mean luminescence intensity of the sample group - mean CC of the blank control) / (mean VC - mean CC)) × 100%. Neutralizing antibody titer is expressed as the reciprocal of the serum dilution corresponding to a 50% inhibition rate or the antibody concentration corresponding to a 50% inhibition rate, defined as the 50% neutralizing antibody titer (NT50). Serum dilutions were serially diluted starting at 1:500. When 100 μl of serum was incubated with 50 μl of pseudovirus, all dilution factors should be multiplied by 1.5 to calculate the neutralizing titer.

[0120] Experimental conclusion: Through intramuscular injection of CLAN@ DOTAP / MC3 / PEG-PLGA 1-55-49 The mRNA combination nanoparticle vaccine induced the expression of S protein-specific IgG antibodies at both 10 μg and 30 μg doses, with the IgG antibody level in the 30 μg / mouse dose group being 7.38 times higher than that in the 10 μg / mouse dose group ( Figure 6 A), and the results of the pseudovirus detection system based on vesicular stomatitis virus (VSV) on the reaction of inactivated serum of mice 42 days after vaccination showed that: CLAN@ DOTAP / MC3 / PEG-PLGA 1-55- 49 The neutralizing antibody activity in the serum of mice vaccinated with mRNA combination nanoparticles showed a dose-dependent manner, with the neutralizing antibody activity in the 30 μg / mouse dose group being 3.37 times higher than that in the 10 μg / mouse dose group ( Figure 6 B).

[0121] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A combination nanoparticle, characterized in that: It is prepared from polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer, cationic lipid, and ionizable lipid, wherein the mass proportion of the cationic lipid is 0.5%-3%, the mass proportion of the ionizable lipid is 50%-60%, the mass proportion of the polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer is 38%-49%, and the total amount of the polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer, cationic lipid, and ionizable lipid is 100%; The ionizable lipids are Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, BHEM-DBA: , BHEM-APMP: , BHEM-EAA: , or BHEM-AEA: ; The cationic lipid is trifluoroacetic acid dimethyl-2,3-dioleyloxypropyl-2-(2-spermine formamido)ethylammonium, trimethyl dodecyl ammonium bromide, trimethyl-2,3-dioleyloxypropyl ammonium bromide, brom(2,3-dioleyloxypropyl)trimethylammonium chloride, trimethyl tetradecyl ammonium bromide, trimethyl hexadecyl ammonium bromide, dimethyl dioctadecyl ammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropyl ammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropyl ammonium bromide, any one of dioleyloxypropylammonium bromide, dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide, dimethyl-4-hydroxybutyl-2,3-dioleyloxypropylammonium bromide, dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dicetadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dicetadecyloxypropylammonium bromide, N-(2-sperminocarbonyl)-N', N'-dioctadecylglycinamide, 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol, lipid poly-L-lysine, and stearylamine.

2. The combined nanoparticles according to claim 1, characterized in that The mass proportion of the cationic lipid is 0.5%-2%.

3. The combined nanoparticles according to claim 2, characterized in that The mass proportion of the cationic lipid is 0.5%-1.5%.

4. The combined nanoparticles according to claim 1, characterized in that The mass proportion of the ionizable lipids is 52% to 56%.

5. The combined nanoparticles according to claim 4, characterized in that The mass proportion of the ionizable lipids is 54% to 56%.

6. The combined nanoparticles according to claim 1, characterized in that The polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid-glycolic acid) copolymer accounts for 35%-46% in the combined nanoparticles.

7. The combined nanoparticles according to claim 6, characterized in that The polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid-glycolic acid) copolymer accounts for 40%-46% in the combined nanoparticles.

8. The combined nanoparticles according to claim 1, characterized in that The polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer is polyethylene glycol-modified polylactide or polyethylene glycol-modified poly(glycolide-co-lactide).

9. The combined nanoparticles according to claim 8, characterized in that The molecular weight of the polyethylene glycol-modified poly(glycolide-co-lactide) is 2k-5k.

10. Use of the combined nanoparticles according to any one of claims 1 to 9 as carriers of nucleic acid drugs in the preparation of combined nanoparticle preparations for preventing and / or treating tumors or viral infections.

11. A combined nanoparticle preparation, characterized in that: The composite nanoparticle is prepared from the composite nanoparticle according to any one of claims 1 to 9 and nucleic acid, and the mass ratio of the composite nanoparticle to the nucleic acid is 125-500:

1.

12. The combined nanoparticle preparation according to claim 11, characterized in that The mass ratio of the combined nanoparticles to the nucleic acid is 200-400:

1.

13. The combined nanoparticle preparation according to claim 11, characterized in that The mass ratio of the combined nanoparticles to the nucleic acid is 220-300:

1.

14. The combined nanoparticle preparation according to claim 11, characterized in that The nucleic acid is siRNA or mRNA.

15. The combined nanoparticle preparation according to claim 14, characterized in that The siRNA or mRNA is a tumor treatment drug or an antiviral infection treatment drug.

16. The method for preparing the combined nanoparticle preparation according to any one of claims 11 to 15, characterized in that: The following steps are involved: S1. Polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-co-glycolic acid) copolymer, a cationic lipid, and an ionizable lipid are dissolved in organic solvents to obtain three solutions; S2. The three solutions were added to ultrapure water, heated to 55°C-70°C, and stirred to prepare composite nanoparticles; S3. Filtration, concentration, and ultrafiltration to obtain a suspension of combined nanoparticles from which the organic solvent has been removed; S4. Mix the nucleic acid aqueous solution with the combined nanoparticle suspension of step S3, and incubate at room temperature to obtain a combined nanoparticle preparation.

17. Use of the combined nanoparticle preparation according to any one of claims 11 to 15 in the preparation of nucleic acid drugs for preventing and / or treating tumors or viral infections.

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