Lipid nanoparticle loaded with mRNA (messenger ribonucleic acid) nano antibody as well as preparation method and application of lipid nanoparticle

Abstract

The invention relates to a lipid nanoparticle loaded with a nano antibody mRNA, the lipid nanoparticle contains a lipid mixture and the nano antibody mRNA, the N / P value of the lipid mixture and the nano antibody mRNA is (3-10): 1, the amino acid sequence of the nano antibody is as shown in SEQ ID NO: 1, and the nucleotide sequence of the nano antibody mRNA is as shown in SEQ ID NO: 2. The nano-antibody mRNA obtained by in-vitro transcription is entrapped in the lipid nanoparticles, so that the nano-antibody can be expressed in living cells, and the prepared lipid nanoparticles loaded with the nano-antibody mRNA have the advantages of high targeting, long half-life period, high entrapment efficiency, good stability, safety, effectiveness and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a lipid nanoparticle loaded with nanobody mRNA, a preparation method thereof, and an application thereof. Background Art

[0002] Nanobodies are variable domains of natural heavy-chain antibodies that are abundantly present in the sera of camelids and sharks. With a molecular weight of only 15 kDa, they are 1 / 10 of the molecular weight of traditional antibodies and are currently the smallest known natural antibodies. Compared with traditional antibodies, nanobodies have a small molecular weight and low immunogenicity, and can be rapidly produced into antiviral preparations for passive immunization. Nanobodies also have good biochemical properties, including high thermal stability and deep tissue permeability. The disadvantage is that they are rapidly cleared in the blood, resulting in too short a half-life of the drug. By using mRNA encoding nanobodies, transient expression can occur in vivo, generating more proteins during peak expression, increasing serum antibody concentration, and prolonging the half-life. However, in vitro transcribed mRNA still faces technical problems such as poor stability and low delivery efficiency.

[0003] Lipid Nanoparticles (LNPs) are the most mainstream mRNA delivery systems. They mainly form complexes by the binding of ionizable cationic lipids to negatively charged mRNA, and then enter cells by endocytosis. In addition to protecting mRNA from degradation, LNPs can also promote cell uptake and improve endosomal escape, thereby enhancing the expression of the target protein and its effect. Summary of the Invention

[0004] The object of the present invention is to provide a lipid nanoparticle loaded with nanobody mRNA. The lipid nanoparticle contains a lipid mixture and nanobody mRNA. Among them, the N / P value of the lipid mixture and nanobody mRNA is 3 - 10:1. The amino acid sequence of the nanobody is as shown in SEQ ID NO: 1, and the nucleotide sequence of the nanobody mRNA is as shown in SEQ ID NO: 2.

[0005] In a preferred technical solution of the present invention, the N / P value of the lipid mixture and nanobody mRNA is 3 - 8:1, preferably 4 - 6:1.

[0006] In a preferred technical solution of the present invention, the lipid mixture is composed of an ionizable lipid, a helper lipid, cholesterol or a cholesterol derivative, and a polyethylene glycol lipid in a molar ratio of 20 - 50:20 - 50:5 - 40:0.5 - 5, preferably a molar ratio of 25 - 50:20 - 45:10 - 40:1 - 2.

[0007] In a preferred technical solution of the present invention, the ionizable lipid is selected from any one or a combination of 9-(heptadecan-9-yl) 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), bis(2-hexyldecanoic acid) bis(hexane-6,1-diyl) bis(4-hydroxybutyl) azodicarbamate) (ALC-0315), chloro-1,2-dilinoleoyl methylammonium (DLinDMAC), 1,2-dilinoleoyl dimethylammonium (DLinDMA), 1,2-dilinoleoyl octadecylammonium (DLinDKA).

[0008] In a preferred technical solution of the present invention, the helper lipid is selected from any one or a combination of distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylcholine (DPPC).

[0009] In a preferred technical solution of the present invention, the cholesterol derivative is selected from any one or a combination of cholesteryl hexanoate, cholesteryl succinate, cholesteryl sulfate, hydroxycholesterol, β-sitosterol, oxidized cholesterol.

[0010] In a preferred technical solution of the present invention, the polyethylene glycol lipid is selected from any one or a combination of dimyristoyl glycerol-polyethylene glycol (DMG-PEG), methoxypolyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG), methoxypolyethylene glycol-dioleoyl phosphatidylethanolamine (DMPE-PEG), methoxypolyethylene glycol-dioleoyl phosphatidylethanolamine (DOPE-mPEG), methoxypolyethylene glycol-dioleoyl phosphatidylcholine (DOPC-mPEG).

[0011] In a preferred technical solution of the present invention, the polyethylene glycol lipid is selected from any one of dimyristoyl glycerol-polyethylene glycol 1000 (DMG-PEG1000), dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000), dimyristoyl glycerol-polyethylene glycol 3400 (DMG-PEG3400), dimyristoyl glycerol-polyethylene glycol 5000 (DMG-PEG5000).

[0012] In a preferred technical solution of the present invention, the lipid mixture is composed of SM-102, cholesterol, DSPC and DMG-PEG.

[0013] In a preferred technical solution of the present invention, the lipid mixture is composed of SM-102, cholesterol, DSPC and DMG-PEG in a molar ratio of 20-50:20-50:5-40:0.5-5, preferably a molar ratio of 25-50:25-45:20-40:1-2.

[0014] In a preferred technical solution of the present invention, the particle size of the lipid nanoparticles is 50 - 300 nm, preferably 120 - 180 nm.

[0015] In a preferred technical solution of the present invention, the PDI of the lipid nanoparticles is ≤ 0.3, preferably PDI ≤ 0.2.

[0016] In a preferred technical solution of the present invention, the Zeta potential of the lipid nanoparticles is -20 - 20 mV, preferably -5 - 5 mV.

[0017] In a preferred technical solution of the present invention, the encapsulation efficiency of the lipid nanoparticles is ≥ 80%, preferably ≥ 90%, more preferably ≥ 95%.

[0018] Another object of the present invention is to provide a method for preparing lipid nanoparticles loaded with nanobody mRNA. The lipid nanoparticles contain a lipid mixture and nanobody mRNA. Among them, the N / P value of the lipid mixture and nanobody mRNA is 3 - 10:1. The amino acid sequence of the nanobody is as shown in SEQ ID NO: 1, and the nucleotide sequence of the nanobody mRNA is as shown in SEQ ID NO: 2. The method for preparing the lipid nanoparticles includes the following steps:

[0019] (1) Dissolve the lipid mixture in ethanol to prepare a lipid mixture ethanol solution, and dissolve the nanobody mRNA in a buffer solution to prepare an mRNA buffer solution. The N / P value of the lipid mixture and nanobody mRNA is 3 - 10:1;

[0020] (2) Add the lipid mixture ethanol solution and the mRNA buffer solution to a microfluidic mixer for mixing to obtain a crude lipid nanoparticle solution. The volume ratio of the lipid ethanol solution to the mRNA buffer solution is 1:1 - 4;

[0021] (3) Dilute the crude lipid nanoparticle solution with phosphate buffer (PBS), place it in an ultrafiltration tube, centrifuge at 4 - 10 °C and 3000 - 5000 rpm, then elute the filtrate with phosphate buffer (PBS) and concentrate it to 1 / 10 - 1 / 2 of the original volume, and filter with a filter membrane to obtain the product.

[0022] In a preferred technical solution of the present invention, in step (1), the buffer solution is any one or a combination of citrate buffer solution, sodium acetate - acetic acid buffer solution, and citrate - phosphate buffer solution.

[0023] In a preferred technical solution of the present invention, in step (1), the buffer solution is a citrate buffer solution of 100 - 400 mM, preferably a citrate buffer solution of 200 - 300 mM.

[0024] In a preferred technical solution of the present invention, in step (1), the pH of the buffer solution is 4-5.

[0025] In a preferred technical solution of the present invention, in step (2), the mixing conditions are as follows: the sum of the flow rates of the lipid mixture ethanol solution and the mRNA buffer solution is 8-20 mL / min, and the flow rate ratio of the lipid mixture ethanol solution to the mRNA buffer solution is 1:1-3.

[0026] In a preferred technical solution of the present invention, in step (3), the centrifugation conditions are centrifugation at 3000-5000 rpm for 5-30 min, preferably centrifugation at 3000-4000 rpm for 10-15 min.

[0027] In a preferred technical solution of the present invention, in step (3), the pH of the phosphate buffer solution (PBS) is 7-7.5.

[0028] In a preferred technical solution of the present invention, in step (3), the ultrafiltration tube is 100 kDa.

[0029] In a preferred technical solution of the present invention, in step (3), the filter membrane is 0.22 μm.

[0030] In a preferred technical solution of the present invention, the N / P value of the lipid mixture and the nanobody mRNA is 3-8:1, preferably 4-6:1.

[0031] In a preferred technical solution of the present invention, the lipid mixture is composed of an ionizable lipid, a helper lipid, cholesterol or a cholesterol derivative, and a polyethylene glycol lipid in a molar ratio of 20-50:20-50:5-40:0.5-5, preferably a molar ratio of 25-50:20-45:10-40:1-2.

[0032] In a preferred technical solution of the present invention, the ionizable lipid is selected from any one or a combination of 9-(1,1-dimethylheptyl) nonyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), bis(2-hexyldecanoic acid) bis(hexane-6,1-diyl) bis(4-hydroxybutyl) azodicarboxylate (ALC-0315), chloro-1,2-linoleoyl-3-dimethylammonium propane (DLinDMAC), 1,2-dilinoleoyl-3-dimethylammonium propane (DLinDMA), 1,2-dilinoleoyl-3-octadecylammonium propane (DLinDKA).

[0033] In a preferred technical solution of the present invention, the helper lipid is selected from any one or a combination of dipalmitoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC).

[0034] In a preferred technical solution of the present invention, the cholesterol derivative is selected from any one or a combination of cholesteryl caproate, cholesteryl succinate, cholesteryl sulfate, hydroxycholesterol, β-sitosterol, and oxidized cholesterol.

[0035] In a preferred technical solution of the present invention, the polyethylene glycol lipid is selected from any one or a combination of dimyristoyl glycerol-polyethylene glycol (DMG-PEG), methoxypolyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG), methoxypolyethylene glycol-dioleoyl phosphatidylethanolamine (DMPE-PEG), methoxypolyethylene glycol-dioleoyl phosphatidylethanolamine (DOPE-mPEG), and methoxypolyethylene glycol-dioleoyl phosphatidylcholine (DOPC-mPEG).

[0036] In a preferred technical solution of the present invention, the polyethylene glycol lipid is selected from any one of dimyristoyl glycerol-polyethylene glycol 1000 (DMG-PEG1000), dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000), dimyristoyl glycerol-polyethylene glycol 3400 (DMG-PEG3400), and dimyristoyl glycerol-polyethylene glycol 5000 (DMG-PEG5000).

[0037] In a preferred technical solution of the present invention, the lipid mixture is composed of SM-102, cholesterol, DSPC, and DMG-PEG.

[0038] In a preferred technical solution of the present invention, the lipid mixture is composed of SM-102, cholesterol, DSPC, and DMG-PEG in a molar ratio of 20-50:20-50:5-40:0.5-5, preferably a molar ratio of 25-50:25-45:20-40:1-2.

[0039] In a preferred technical solution of the present invention, the particle size of the lipid nanoparticles is 50-300 nm, preferably 120-180 nm.

[0040] In a preferred technical solution of the present invention, the PDI of the lipid nanoparticles is ≤0.3, preferably PDI ≤0.2.

[0041] In a preferred technical solution of the present invention, the Zeta potential of the lipid nanoparticles is -20 - 20 mV, preferably -5 - 5 mV.

[0042] In a preferred technical solution of the present invention, the encapsulation efficiency of the lipid nanoparticles is ≥80%, preferably ≥90%, more preferably ≥95%.

[0043] Another object of the present invention is to provide a pharmaceutical composition, which is composed of the lipid nanoparticles loaded with nanobody mRNA of the present invention and a pharmaceutically acceptable carrier.

[0044] In a preferred technical solution of the present invention, the pharmaceutically acceptable carrier of the present invention includes any one or a combination of a pH regulator, an osmotic pressure regulator, and a lyoprotectant.

[0045] In a preferred technical solution of the present invention, the pH regulator is selected from any one or a combination of sodium citrate, potassium citrate, malic acid, sodium malate, potassium malate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium acetate, Tris-HCl, and histidine buffer solution.

[0046] In a preferred technical solution of the present invention, the osmotic pressure regulator is selected from any one of sodium chloride, potassium chloride, glucose, phosphate, citrate, and mannose.

[0047] In a preferred technical solution of the present invention, the lyoprotectant is selected from any one or a combination of sucrose, trehalose, mannitol, and silk fibroin.

[0048] In a preferred technical solution of the present invention, the administration method of the pharmaceutical composition is any one of intravenous administration, intramuscular injection, nasal administration, inhalation administration, and subcutaneous administration.

[0049] Another object of the present invention is to provide the use of lipid nanoparticles loaded with nanobody mRNA in the preparation of a drug for preventing and treating respiratory virus infections.

[0050] In a preferred technical solution of the present invention, the respiratory virus includes any one of influenza virus, coronavirus, respiratory syncytial virus, adenovirus, and rhinovirus.

[0051] In a preferred technical solution of the present invention, the coronavirus is selected from any one or a variant strain of SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKΜ1, SARS-CoV, and MERS-CoV.

[0052] Unless otherwise specified, the N / P ratio in the present invention is the molar ratio of ammonium (N) on the ionizable cationic lipid in the lipid mixture to anionic phosphate (P) in the nanobody mRNA.

[0053] The nanobody mRNA in the present invention is a nucleic acid sequence encoding a nanobody, and the sequence is as shown in SEQ ID NO: 2.

[0054] The present invention adopts the following detection methods:

[0055] 1. Use a Malvern nanoparticle size analyzer to detect the particle size, Zeta potential, and particle size distribution index (PDI) of the lipid nanoparticles.

[0056] 2. Standard curve: Take an appropriate amount of 20×TE buffer solution and dilute it to 1×TE buffer solution with DEPC water. Accurately measure a certain volume of RNA standard solution (100 μg / mL) and dilute it to 2000 ng / mL with 1×TE buffer solution. Add 2000 ng / mL RNA standard solution to a black 96-well plate at 0, 1, 2, 10, 25, 50, 75, 100 μL, and make up to 100 μL with 1×TE buffer solution to prepare RNA standard solutions with concentrations of 0, 20, 40, 200, 500, 1000, 1500, 2000 ng / mL, and prepare three parallel samples. Dilute RiboGreen dye according to a 1:200 ratio with 1×TE buffer solution to prepare RiboGreen working solution. Add an equal volume of RiboGreen working solution (i.e., 100 μL) to the RNA standard solution wells to make the final concentrations of the RNA standard solutions 0, 10, 20, 100, 250, 500, 750, 1000 ng / mL for measuring the concentration of free RNA. Take an appropriate amount of TritonX-100 solution and dilute it to 0.5% Triton X-100 solution with 1×TE buffer solution. Dilute the RNA standard solution with 0.5% Triton X-100 solution and operate according to the above steps to prepare samples for measuring the total RNA concentration.

[0057] 3. Determination of encapsulation efficiency sample: Take 1 μL of sample solution and add it to a black 96-well plate, add 99 μL of 1×TE buffer solution, add 100 μL of RiboGreen staining working solution, incubate at room temperature in the dark for 5 min, use a microplate reader, select the fluorescence mode, set Ex = 480 nm, Em = 532 nm, measure its RFU, and calculate the content of free mRNA in the sample. Treat the sample with 0.5% TritonX-100 solution in the same way, calculate the total mRNA content in the sample, and calculate the encapsulation efficiency.

[0058] 4. Non-formaldehyde denaturing agarose gel electrophoresis experiment: According to the mRNA loading amount of 100 ng, take an appropriate amount of lipid nanoparticle sample solution; mix 2×RNA Loading Dye and lipid nanoparticle sample solution evenly at a ratio of 1:1; mix 3 μL of ssRNA Ladder and 7 μL of 2×RNA Loading Dye evenly; incubate the above samples in a PCR instrument at 65 °C for 10 min, immediately put them on ice, ice bath for 2 min, add them to the sample wells after ice bath, turn on the power supply, 180 V, and electrophoresis for 20 min.

[0059] 5. Detection of IgG antibody titer in mouse serum by ELISA method: Dilute the 2.5 mg / mL RBD protein solution (Sino Biological) to 1 μg / mL with 1× coating buffer, add 100 μL per well to a high-affinity ELISA plate with 96 wells, seal the plate with a sealing film, and incubate overnight at 4°C; discard the coating solution, wash the plate 3 times with 200 μL of 1× PBST, block with 1× PBST solution containing 2% bovine serum albumin (BSA), add 200 μL of blocking buffer per well, seal the plate with a sealing film, and block at 37°C for 1 h; discard the blocking solution, wash the plate 3 times with 200 μL of 1× PBST per well, serially dilute the immune serum in the 96-well plate with a dilution solution (1× PBST solution containing 0.1% BSA) starting from 1:300 with a 3-fold serial dilution, 100 μL per well, and the sample well without serum is the control group, seal the plate with a sealing film, and incubate at 37°C for 2 h; discard the sample, wash the plate 5 times with 200

[0060] μL of 1× PBST per well, add HRP-labeled goat anti-human (H+L) HRP (1:2000) diluted with the dilution solution, 100 μL per well, seal the plate with a sealing film, and incubate at 37°C for 1 h; discard the sample, wash the plate 5 times with 200 μL of 1× PBST per well, add 200 μL of chromogenic agent TMB per well, seal the plate with a sealing film, and incubate at 37°C in the dark for 15 min; after the incubation ends (the solution in the ELISA plate gradually changes from colorless to blue), add 50 μL of ELISA stop solution per well, and immediately measure the absorbance (Optical density, OD) value at 450 nm using an ELISA reader; measure the OD value at 450 nm using an ELISA reader, where the OD value higher than 2.1 times that of the control group is positive, and its maximum dilution multiple is the titer value of the specific antibody in the serum of this sample.

[0061] Compared with the prior art, the present invention has the following beneficial technical effects:

[0062] 1. The present invention encapsulates the in vitro transcribed nanobody mRNA in lipid nanoparticles; the prepared lipid nanoparticles loaded with nanobody mRNA can express nanobodies in living cells, and have the advantages of high targeting, long half-life, high encapsulation efficiency, good stability, safety and effectiveness, etc.

[0063] 2. The preparation process of the present invention is simple and low-cost, and can be used for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Agarose gel electrophoresis diagram of the nanobody mRNA of the present invention;

[0065] Figure 2 Schematic diagram of the preparation of lipid nanoparticles loaded with nanobody mRNA of the present invention;

[0066] Figure 3 Gel electrophoresis pattern of the lipid nanoparticles loaded with nanobody mRNA of the present invention;

[0067] Figure 4 Cell transfection effect diagram of the lipid nanoparticles loaded with nanobody mRNA of the present invention;

[0068] Figure 5 Stability results of the lipid nanoparticles loaded with nanobody mRNA of the present invention;

[0069] Figure 6 Half-life study of the lipid nanoparticles loaded with nanobody mRNA of the present invention;

[0070] Figure 7 Safety evaluation results of the lipid nanoparticles loaded with nanobody mRNA of the present invention. Detailed implementation manners

[0071] The present invention will be described below with reference to the examples. However, the present invention is not limited to the examples.

[0072] Marker (i.e., ssRNA Ladder) was commercially purchased from NEB Company, USA (product number: 10156802). The microfluidic mixer (commercially purchased from Shanghai Pengzan Biotechnology Co., Ltd., model LNP-S1).

[0073] Reference example Preparation of nanobody mRNA of the present invention

[0074] 1. Sequence design

[0075] The VHH 72 nanobody (GenBank serial number on NCBI: QJG66105.1) was selected, and the amino acid sequence of the nanobody is shown in SEQ ID NO: 1:

[0076] QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSS

[0077] The T7-mediated in vitro transcription vector was selected to improve the translation efficiency, and its codons were humanized and modified, and the coding nucleic acid sequence was redesigned, and the sequence is shown in SEQ ID NO: 2:

[0078] CAGGUGCAGC UGCAGGAGUC UGGGGGAGGA UUGGUGCAGG CUGGGGGCUC UCUGAGACUC

[0079] UCCUGUGCCG CCUCUGGACG CACCUUCAGU GAAUAUGCCA UGGGCUGGUU CCGCCAGGCU

[0080] CCAGGGAAGG AACGUGAGUU UGUAGCAACU AUUAGCUGGA GUGGUGGUAG CACAUACUAU

[0081] ACAGACUCCG UGAAGGGCCG AUUCACCAUC UCCAGAGACA ACGCCAAGAA CACGGUGUAU

[0082] CUGCAAAUGA ACAGCCUGAA ACCUGAUGAC ACGGCCGUUU AUUACUGUGC AGCAGCCGGG

[0083] UUAGGUACGG UAGUAUCGGA GUGGGAUUAU GACUAUGACU ACUGGGGCCA GGGGACCCAG

[0084] GUCACCGUCU CCUCA。

[0085] It was commissioned to Yunzhou Biotechnology Co., Ltd. for synthesis to obtain a recombinant plasmid encoding the nanobody.

[0086] 2. Plasmid extraction and in vitro transcription

[0087] Add 125 μL of kanamycin solution (0.05%, working concentration 50 μg / mL) to the LB liquid medium. Inoculate the Escherichia coli bacterial solution containing the recombinant plasmid encoding the nanobody in step (1) according to the ratio of the bacterial solution to the medium of 1:1000 - 1:500 (v / v). Seal it with a sterile sealing film and place it in a constant temperature shaker at 37°C, 180 - 220 rpm, and culture for 12 - 16 h. Use the The plasmid solution was extracted using the EndoFree Plasmid Maxi Kit. Then, the plasmid was digested with the SapI restriction endonuclease kit from NEB. Samples were prepared according to the system and placed in 1.5 mL centrifuge tubes. The reaction was carried out overnight at 37 °C for 12 - 16 h. The linear DNA was purified using the phenol extraction method. Subsequently, an in vitro transcription experiment was performed using the T7 High Yield RNA Transcription kit from Novoprotein. The components were gently mixed with a pipette and briefly centrifuged to collect. The mixture was incubated in a PCR instrument at 37 °C for 3 h. The capping reaction was carried out according to the Cap 1 Capping System kit from Novoprotein. The reaction system was prepared and reacted at 37 °C for 30 min. Lithium chloride solution and DEPC water were added, and it was placed at -20 °C for 1 hour. After centrifugation, the supernatant was removed, and the precipitate was collected. The mRNA precipitate was rinsed with 70% ethanol solution, air-dried, and then dissolved in DEPC to obtain the nanobody mRNA solution, and its concentration was detected to be 1 μg / μL.

[0088] 3. Agarose gel electrophoresis

[0089] The nanobody mRNA solution was taken for agarose gel electrophoresis. The samples were run on the gel with a loading amount of 500 ng mRNA per well. Observation and photography were carried out at 302 nm ultraviolet using a gel imager. The results are shown in Figure 1 . The bands were bright and clear, the number of bases was consistent with the designed mRNA base number, and the integrity of the mRNA was good.

[0090] Example 1 Preparation of lipid nanoparticles loaded with nanobody mRNA of the present invention

[0091] The schematic diagram of the preparation of lipid nanoparticles loaded with nanobody mRNA of the present invention is shown in Figure 2 , and specifically includes the following steps:

[0092] (1) SM-102, DSPC, cholesterol, and DMG-PEG2000 were dissolved in absolute ethanol respectively to prepare 20 mg / mL SM-102 ethanol solution, 20 mg / mL DSPC ethanol solution, 20 mg / mL cholesterol ethanol solution, and 20 mg / mL DMG-PEG2000 ethanol solution. The four ethanol solutions were mixed according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 33:25.5:40:1.5 to obtain a lipid mixture ethanol solution, and absolute ethanol was added to dilute to a molar concentration of 8 mM for the lipid mixture;

[0093] (2) According to the N:P ratio of lipid mixture and nanobody mRNA being 3:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example, dissolve it in 300 mM citrate buffer (pH = 4) to prepare the mRNA nanobody buffer solution with the required concentration;

[0094] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer for mixing. The flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, and the total flow rate of the two is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0095] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five times the volume of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute with five times the volume of PBS solution and concentrate to 1 / 2 of the original volume, and filter with a 0.22 μm filter membrane to obtain.

[0096] According to the method of the present invention, the encapsulation efficiency of the lipid nanoparticles loaded with nanobody mRNA is 83.28%, the particle size is 174.75 nm, the Zeta potential is 4.2 mV, and the PDI is 0.24.

[0097] Example 2 Preparation of lipid nanoparticles loaded with nanobody mRNA of the present invention

[0098] The preparation of the lipid nanoparticles loaded with nanobody mRNA of the present invention includes the following steps:

[0099] (1) Take SM-102, DSPC, cholesterol, and DMG-PEG2000 and dissolve them separately with absolute ethanol to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution; mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 being 33:25.5:40:1.5 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol to dilute to a molar concentration of 8 mM of the lipid mixture;

[0100] (2) According to the N:P ratio of lipid mixture and nanobody mRNA being 4:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example, dissolve it in 300 mM citrate buffer (pH = 4) to prepare the mRNA nanobody buffer solution with the required concentration;

[0101] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer for mixing. The flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, and the total flow rate of the two is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0102] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five times its volume of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute with five times the volume of PBS solution and concentrate to 1 / 2 of the original volume, and filter through a 0.22 μm filter membrane to obtain the product.

[0103] According to the method of the present invention, the encapsulation efficiency of the lipid nanoparticles loaded with nanobody mRNA is 89.03%, the particle size is 146.87 nm, the Zeta potential is 8.6 mV, and the PDI is 0.18.

[0104] Example 3 Preparation of lipid nanoparticles loaded with nanobody mRNA of the present invention

[0105] The preparation of the lipid nanoparticles loaded with nanobody mRNA of the present invention includes the following steps:

[0106] (1) Dissolve SM-102, DSPC, cholesterol, and DMG-PEG2000 in absolute ethanol respectively to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution; mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 33:25.5:40:1.5 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol to dilute to a molar concentration of 8 mM of the lipid mixture;

[0107] (2) According to the N:P ratio of the lipid mixture to nanobody mRNA of 6:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example and dissolve it in 300 mM citrate buffer (pH = 4) to prepare the required concentration of mRNA nanobody buffer solution;

[0108] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer for mixing. The flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, and the total flow rate of the two is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0109] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five volumes of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute and concentrate with five volumes of PBS solution to 1 / 2 of the original volume, and filter through a 0.22 μm filter membrane to obtain the product.

[0110] According to the method of the present invention, the encapsulation efficiency of the lipid nanoparticles loaded with nanobody mRNA is 91.86%, the particle size is 130.03 nm, the Zeta potential is 11.2 mV, and the PDI is 0.263.

[0111] Example 4 Preparation of the nanobody mRNA lipid nanoparticles of the present invention

[0112] The preparation of the nanobody mRNA lipid nanoparticles of the present invention includes the following steps:

[0113] (1) Take SM-102, DSPC, cholesterol, and DMG-PEG2000 and dissolve them separately in absolute ethanol to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution; mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 33:25.5:40:1.5 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol to dilute to a molar concentration of 8 mM of the lipid mixture;

[0114] (2) According to the N:P ratio of the lipid mixture and nanobody mRNA of 8:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example and dissolve it in 300 mM citrate buffer (pH = 4) to prepare the required concentration of mRNA nanobody buffer solution;

[0115] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer for mixing, and the flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, and the total flow rate of the two is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0116] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five volumes of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute and concentrate with five volumes of PBS solution to 1 / 2 of the original volume, and filter through a 0.22 μm filter membrane to obtain the product.

[0117] The encapsulation efficiency of the lipid nanoparticles loaded with nanobody mRNA detected by the method of the present invention is 90%, the particle size is 155.73 nm, the Zeta potential is 7.9 mV, and the PDI is 0.16.

[0118] Example 5 Preparation of lipid nanoparticles loaded with nanobody mRNA of the present invention

[0119] The preparation of the lipid nanoparticles loaded with nanobody mRNA of the present invention includes the following steps:

[0120] (1) Take SM-102, DSPC, cholesterol, and DMG-PEG2000 and dissolve them separately with absolute ethanol to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution; mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 33:25.5:40:1.5 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol to dilute to a molar concentration of 8 mM of the lipid mixture;

[0121] (2) According to the N:P ratio of the lipid mixture and nanobody mRNA of 6:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example and dissolve it in 100 mM citrate buffer (pH = 4) to prepare the mRNA nanobody buffer solution with the required concentration;

[0122] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer for mixing, and the flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, and the total flow rate of the two is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0123] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five times the volume of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute 2 times with five times the volume of PBS solution, concentrate to 1 / 2 of the original volume, and filter with a 0.22 μm filter membrane to obtain.

[0124] Example 6 Preparation of lipid nanoparticles loaded with nanobody mRNA of the present invention

[0125] The preparation of the lipid nanoparticles loaded with nanobody mRNA of the present invention includes the following steps:

[0126] (1) Dissolve SM-102, DSPC, cholesterol, and DMG-PEG2000 separately in absolute ethanol to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution respectively; mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 33:25.5:40:1.5 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol to dilute to a molar concentration of 8 mM for the lipid mixture;

[0127] (2) According to the N:P ratio of the lipid mixture to the nanobody mRNA of 6:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example and dissolve it in 200 mM citrate buffer (pH = 4) to prepare an mRNA nanobody buffer solution with the required concentration;

[0128] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer, and the flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, with a total flow rate of 12 mL / min, to obtain a crude lipid nanoparticle solution;

[0129] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five volumes of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute and concentrate with five volumes of PBS solution to 1 / 2 of the original volume, and filter with a 0.22 μm filter membrane to obtain the product.

[0130] Example 7 Preparation of Lipid Nanoparticles Loaded with Nanobody mRNA

[0131] The preparation of the lipid nanoparticles loaded with nanobody mRNA of the present invention includes the following steps:

[0132] (1) Dissolve SM-102, DSPC, cholesterol, and DMG-PEG2000 separately in absolute ethanol to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution respectively; mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 33:25.5:40:1.5 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol to dilute to a molar concentration of 8 mM for the lipid mixture;

[0133] (2) According to the N:P ratio of lipid mixture and nanobody mRNA being 6:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example, dissolve it in 400 mM citrate buffer (pH = 4) to prepare the mRNA nanobody buffer solution with the required concentration;

[0134] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer for mixing. The flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, and the total flow rate is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0135] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five times its volume of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute twice with five times the volume of PBS solution, concentrate to 1 / 2 of the original volume, and filter through a 0.22 μm filter membrane to obtain the product.

[0136] Comparative Example 1

[0137] The preparation of lipid nanoparticles includes the following steps:

[0138] (1) Take SM-102, DSPC, cholesterol, and DMG-PEG2000 and dissolve them separately in absolute ethanol to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution; Mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 being 33:25.5:40:1 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol for dilution to a molar concentration of 8 mM for the lipid mixture;

[0139] (2) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of 300 mM citrate buffer (pH = 4), add them to a microfluidic mixer for mixing. The flow rate ratio of the lipid mixture ethanol solution to the citrate buffer is 1:3, and the total flow rate of the two is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0140] (3) Dilute the crude lipid nanoparticle solution prepared in step (2) with five times its volume of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute and concentrate to 1 / 2 of the original volume with five times the volume of PBS solution, and filter through a 0.22 μm filter membrane to obtain the product.

[0141] The particle size of the lipid nanoparticles detected by the method of the present invention is 167.96 nm, the Zeta potential is -0.8 mV, and the PDI is 0.38.

[0142] Preparation of lipid nanoparticles loaded with nanobody mRNA in Comparative Example 2

[0143] The preparation of lipid nanoparticles loaded with nanobody mRNA includes the following steps:

[0144] (1) Take SM-102, DSPC, cholesterol, and DMG-PEG2000 and dissolve them separately in absolute ethanol to prepare a 20 mg / mL SM-102 ethanol solution, a 20 mg / mL DSPC ethanol solution, a 20 mg / mL cholesterol ethanol solution, and a 20 mg / mL DMG-PEG2000 ethanol solution; mix the four ethanol solutions according to the molar ratio of SM-102:DSPC:cholesterol:DMG-PEG2000 of 50:38.5:10:1.5 to obtain a lipid mixture ethanol solution, and supplement absolute ethanol to dilute to a molar concentration of 8 mM of the lipid mixture;

[0145] (2) According to the N:P ratio of the lipid mixture and nanobody mRNA of 6:1, take the nanobody mRNA solution with a concentration of 1 μg / μL in the reference example and dissolve it in 25 mM citrate buffer (pH = 4) to prepare an mRNA nanobody buffer solution with the required concentration;

[0146] (3) Take 0.5 mL of the lipid mixture ethanol solution prepared in step (1) and 1.5 mL of the mRNA nanobody buffer solution prepared in step (2), add them to a microfluidic mixer for mixing, and the flow rate ratio of the lipid mixture ethanol solution to the mRNA nanobody buffer solution is 1:3, and the total flow rate of the two is 12 mL / min to obtain a crude lipid nanoparticle solution;

[0147] (4) Dilute the crude lipid nanoparticle solution prepared in step (3) with five times the volume of phosphate buffered saline (PBS, pH 7.4), place it in a 100 kDa ultrafiltration tube, centrifuge at 4 °C and 3000 rpm for 15 min, then elute twice with five times the volume of PBS solution, concentrate to 1 / 2 of the original volume, and filter through a 0.22 μm filter membrane to obtain the product.

[0148] The encapsulation efficiency of the lipid nanoparticles loaded with nanobody mRNA detected by the method of the present invention is 94.31%, the particle size is 129.27 nm, the Zeta potential is -1.48 mV, and the PDI is 0.19.

[0149] Experimental Example 1 Gel electrophoresis analysis and characterization of the lipid nanoparticles loaded with nanobody mRNA of the present invention

[0150] The lipid nanoparticles of Examples 1-4, the lipid nanoparticles of Comparative Examples 1-2, and the nanobody mRNA solution prepared in the reference example were characterized by gel electrophoresis analysis. The results are shown in Figure 3 . There is a bright single band in the mRNA group lane, indicating good integrity of the mRNA. The mRNA in the LNP remains in the sample well, indicating successful loading of the nanobody mRNA.

[0151] Experimental Example 2 Cell transfection experiment of the lipid nanoparticles loaded with nanobody mRNA of the present invention

[0152] An experimental group, a control group, and a blank group were set up. In each group, 293T cells were evenly seeded in a 6-well plate at a concentration of 1×10 6 cells / well and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h. When the cell confluence was about 70%, the cell culture medium was discarded.

[0153] Experimental group: Take a sterile centrifuge tube and add the lipid nanoparticles prepared in Example 3 (containing 2 μg mRNA);

[0154] Control group: Take a sterile centrifuge tube and add the lipid nanoparticles prepared in Comparative Example 2 (containing 2 μg mRNA);

[0155] Blank group: Take a sterile centrifuge tube and add PBS solution;

[0156] Transfer the well plate to the cell culture incubator. After culturing for 6 h, discard the medium in the well plate, add 2 mL of fresh complete DMEM medium (containing 10% FBS and 1% penicillin-streptomycin solution), and continue to culture in the cell culture incubator for 24 h. After the culture is completed, take out the well plate, discard the culture solution, add 1 mL of complete DMEM medium, pipette the cells to make the cell suspension, transfer the cell suspension into a 1.5 mL EP tube, centrifuge at 4°C and 350×g for 5 min, and discard the supernatant; wash once with PBS, centrifuge at 4°C and 350×g for 5 min, and discard the supernatant; resuspend the cells with 0.5 mL of PBS, and evaluate the LNP transfection effect with an Attμne NxT flow cytometer. The results are shown in Figure 4 .

[0157] Compared with the blank group, the lipid nanoparticles of Example 3 and Comparative Example 2 can both transfect cells, indicating that the LNP delivery of nanobody mRNA has a certain promoting effect on cell transfection, so that the nanobody is effectively expressed in target cells. When the dosage is the same, the transfection efficiency of the lipid nanoparticles of Example 3 (86.30%) is better than that of the lipid nanoparticles of Comparative Example 2 (76.30%).

[0158] Experimental Example 3: Study on the Stability of the Lipid Nanoparticles Loaded with Nanobody mRNA of the Present Invention

[0159] Respectively take the lipid nanoparticles of Example 3 (containing 1 μg mRNA), the lipid nanoparticles of Comparative Example 2 (containing 1 μg mRNA), and the reference example nanobody mRNA solution (containing 1 μg mRNA) and incubate them in 50% FBS (fetal bovine serum) at 37 °C for 48 h. Then add 200 μL of a triton X-100 solution with a final concentration of 0.5% and incubate for 5 min to disrupt the LNP structure. Observe the mRNA stability by agarose gel electrophoresis, and the results are shown in Figure 5 . Compared with the mRNA in the lipid nanoparticles of Comparative Example 2, there is obvious degradation or band diffusion, while when the mRNA in Example 3 is incubated with serum, the band is bright and clear, showing higher stability.

[0160] Experimental Example 4: Study on the Half-life of the Lipid Nanoparticles Loaded with Nanobody mRNA of the Present Invention

[0161] Select 9 female mice with a body weight of 18 - 20 g and an age of 6 - 8 weeks, and randomly divide them into 3 groups, with 3 mice in each group:

[0162] mRNA group: Inject 100 μL of a solution obtained by uniformly mixing 95 μL of PBS and 5 μL of the nanobody mRNA solution prepared in the reference example;

[0163] Experimental group: Inject 5 μg of the lipid nanoparticles prepared in Example 3;

[0164] Control group: Inject 5 μg of the lipid nanoparticles prepared in Comparative Example 2;

[0165] After 14 days, use the ELISA method to detect the IgG antibody titer in the mouse serum and study the ability of the protein produced by the serum to bind to RBD. The results are shown in Figure 6 . In the absence of LNP, the level of nanobody drops to about 1% of its initial peak level on the second day, proving that LNP can extend the serum half-life of the antibody, and the level of nanobody in the optimized LNP remains at about 80% of the peak level on the 14th day of the experiment, having the longest antibody serum half-life.

[0166] Experimental Example 5: Safety Evaluation of the Lipid Nanoparticles Loaded with Nanobody mRNA of the Present Invention

[0167] Select 9 female mice with a body weight of 18 - 20 g and an age of 6 - 8 weeks, and randomly divide them into 3 groups, with 3 mice in each group:

[0168] mRNA group: Inject 100 μL of a solution obtained by uniformly mixing 95 μL of PBS and 5 μL of the nanobody mRNA solution prepared in the reference example;

[0169] Experimental group: Inject 5 μg of the lipid nanoparticles prepared in Example 3;

[0170] Control group: Inject 5 μg of the lipid nanoparticles prepared in Comparative Example 2;

[0171] After 1 week, the main organs of the mice, such as the heart, liver, spleen, lungs, and kidneys, were removed and subjected to H&E staining analysis. The results are shown in Figure 7 . Compared with the control group, none of the groups caused toxic pathological damage to the main organs such as the heart, liver, spleen, lungs, and kidneys. The constructed delivery vector has good safety and no obvious adverse reactions.

[0172] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention. As long as they do not depart from the spirit of the present invention, they shall fall within the scope of protection of the claims of the present invention.

Claims

1. A lipid nanoparticle loaded with nano antibody mRNA, the lipid nanoparticle containing a lipid mixture and nano antibody mRNA, wherein: The N / P value of the lipid mixture and the nanobody mRNA is 3-10:1, the amino acid sequence of the nanobody is shown in SEQ ID NO: 1, and the nucleotide sequence of the nanobody mRNA is shown in SEQ ID NO: ID NO:

2.

2. The lipid nanoparticle as claimed in claim 1, wherein the lipid mixture is composed of ionizable lipids, auxiliary lipids, cholesterol or cholesterol derivatives and polyethylene glycol lipids in a molar ratio of 20-50:20-50:5-40:0.5-5, preferably in a molar ratio of 25-50:25- 45:20-40:1-2。 3. The lipid nanoparticles according to any one of claims 1 to 2, wherein the ionizable lipid is selected from heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino)octanoate) (SM-102), bis(2-hexyldecanoate)di(hexane-6,1-diyl)bis(4-hydroxybutyl)azodiether) (ALC-0315), 1,2-linolenic acid dimethylene ammonium chloride (DLinDMAC), 1,2-linolenic acid dilinoleyl dimethyl ammonium (DLinDMA), Any one or a combination of 1,2-dilinoleyldioctadecyl ammonium linolenate (DLinDKA).

4. The lipid nanoparticle of any one of claims 1 to 3, wherein the auxiliary lipid is selected from any one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC) or a combination thereof.

5. The lipid nanoparticle according to any one of claims 1 to 4, wherein the cholesterol derivative is selected from any one of cholesterol hexanoate, cholesterol succinate, cholesterol sulfate hydroxycholesterol, β-sitosterol, oxidized cholesterol or a combination thereof.

6. The lipid nanoparticles of any one of claims 1 to 5, wherein the polyethylene glycol lipid is selected from any one of polyethylene glycol-dimyristoylglycerol (DMG-PEG), methoxy polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG), methoxy polyethylene glycol-dioleoylphosphatidylethanolamine (DMPE-PEG), methoxy polyethylene glycol-dioleoylphosphatidylethanolamine (DOPE-mPEG), and methoxy polyethylene glycol-dioleoylphosphatidylcholine (DOPC-mPEG) or a combination thereof.

7. The lipid nanoparticle of claim 1 , wherein the lipid mixture is composed of SM-102, cholesterol, DSPC and DMG-PEG in a molar ratio of 20-50:20-50:5-40:0.5-5, and a preferred molar ratio of 25-50:25-45:20-40:1-2.

8. A method for preparing lipid nanoparticles loaded with nanobody mRNA as described in any one of claims 1 to 7, comprising the following steps: (1) dissolving the lipid mixture in ethanol to form a lipid mixture ethanol solution, and dissolving the nanobody mRNA in a buffer to form an mRNA buffer, wherein the N / P value of the lipid mixture and the nanobody mRNA is 3-10:1; preferably, the buffer is any one of a citrate buffer, a sodium acetate-acetate buffer, and a citrate-phosphate buffer or a combination thereof; (2) mixing the lipid mixture ethanol solution and the mRNA buffer solution in a microfluidic mixer to obtain a crude lipid nanoparticle solution, wherein the volume ratio of the lipid ethanol solution to the mRNA buffer solution is 1:1-4; (3) The crude lipid nanoparticle solution is diluted with PBS solution, placed in an ultrafiltration tube, and centrifuged at 4°C and 3000-5000 rpm. The filtrate is eluted with PBS solution and concentrated to 1 / 10-1 / 2 of the original volume, and filtered through a filter membrane to obtain the product.

9. A pharmaceutical composition, comprising lipid nanoparticles loaded with nanobody mRNA as described in any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

10. Use of lipid nanoparticles loaded with nanobody mRNA as described in any one of claims 1-7 in the preparation of a medicament for preventing and treating respiratory viral infection, preferably, the respiratory virus includes any one of influenza virus, coronavirus, respiratory syncytial virus, adenovirus, and rhinovirus.

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