A composite lipid nanoparticle, a preparation method thereof, an RNA vaccine, a drug and an application

Abstract

The present invention belongs to the technical field of biological applications of lipid nanoparticles, and discloses composite lipid nanoparticles, a preparation method thereof, an RNA vaccine, a drug and an application. The composite lipid nanoparticles provided by the present invention comprise a lipid composition and a recombinant antibody protein with a streptavidin tag; and the lipid composition comprises an ionizable lipid, a co-lipid, cholesterol, a polyethylene glycol lipid and a biotinylated polyethylene glycol lipid in a mass ratio of (40-90):(0.1-20):(20-50):(0.5-10):(0.5-10). The composite lipid nanoparticles achieve a safe, convenient, fast, efficient and stable connection between the LNP and the recombinant antibody protein through the strong interaction between the recombinant antibody protein with a streptavidin tag in the composite lipid nanoparticles and the biotinylated polyethylene glycol lipid, and the recombinant antibody protein can be adaptively replaced according to the target cells and / or tissues, with high flexibility and applicability, and the recombinant antibody protein on the obtained composite lipid nanoparticles still has good specific recognition and binding ability with the corresponding protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological applications of lipid nanoparticles, and particularly relates to a composite lipid nanoparticle, a preparation method thereof, an RNA vaccine, a drug and an application. Background Art

[0002] Lipid Nanoparticles (LNP) are a novel nanotechnology product, usually composed of four components: phospholipid lipids, cholesterol, polymer-modified lipids, and ionic lipids. The above-mentioned components together form a stable bilayer structure similar to the cell membrane. LNP can encapsulate and protect mRNA, and at the same time, by connecting ligand proteins at the target site on the LNP, it helps mRNA and drugs to accurately reach and act on target cells or tissues, and the materials used in LNP can be safely decomposed in the body, so it is widely used in the field of nucleic acid and drug delivery.

[0003] Currently, the connection between LNP and protein is usually achieved by covalent connection. According to the reaction mechanism, it can be specifically divided into maleimide-thiol reaction method, click chemistry method, and amino-carboxyl crosslinking method. Among them, the maleimide-thiol reaction method is to introduce maleimide groups on the surface of LNP, and use the maleimide group to form a stable covalent bond with the thiol (-SH) naturally present or engineered into the protein to achieve stable connection of the protein. The click chemistry method is to introduce alkynyl and azide groups on LNP and protein respectively, and quickly form a covalent bond through azide-alkyne click chemistry. The amino-carboxyl crosslinking method activates the carboxyl group on the surface of LNP or the amino group on the protein, and uses EDC or NHS for coupling reaction to form an amide bond between LNP and the protein to achieve connection.

[0004] However, the above-mentioned covalent connection has problems such as poor stability, non-specific connection, low connection efficiency, chemical reagent residue, and high requirements for operating conditions, and has great limitations. Summary of the Invention

[0005] The first object of the present invention is to solve the problems existing in the existing methods for realizing the connection between LNP and protein, such as poor stability, non-specific connection, low connection efficiency, chemical reagent residue, high requirements for operating conditions, etc. Therefore, a composite lipid nanoparticle is provided. Through the strong interaction between the recombinant antibody protein with streptavidin tag in the composite lipid nanoparticle and the biotinylated polyethylene glycol lipid, the safe, convenient, fast, efficient and stable connection between LNP and the recombinant antibody protein can be realized, and the recombinant antibody protein can be adaptively replaced according to the target cells and / or tissues, with high flexibility and applicability. Moreover, the recombinant antibody protein on the obtained composite lipid nanoparticle still has good specific recognition and binding ability with the corresponding protein.

[0006] The second object of the present invention is to provide a preparation method of the above composite lipid nanoparticle.

[0007] The third object of the present invention is to provide an RNA vaccine.

[0008] The fourth object of the present invention is to provide a drug.

[0009] The fifth object of the present invention is to provide the application of the above-mentioned composite lipid nanoparticle in the fields of nucleic acid and drug delivery.

[0010] Specifically, the composite lipid nanoparticle provided by the present invention includes: a lipid composition and a recombinant antibody protein with a streptavidin tag; the lipid composition includes an ionizable lipid, a co-lipid, cholesterol, a polyethylene glycol lipid and a biotinylated polyethylene glycol lipid, and the mass ratio of the ionizable lipid, the co-lipid, cholesterol, the polyethylene glycol lipid and the biotinylated polyethylene glycol lipid is (40-90):(0.1-20):(20-50):(0.5-10):(0.5-10).

[0011] In some specific embodiments, the ionizable lipid is selected from one or more of trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleenyloxypropylammonium chloride, 3β-[N-(N’,N’-dimethylaminoethyl)carbamoyl] and 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate.

[0012] In some specific embodiments, the co-lipid is selected from one or more of dioleoyl phosphatidylethanolamine, stearoyl phosphatidylcholine, distearoyl phosphatidylcholine and sterol.

[0013] In some specific embodiments, the polyethylene glycol lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol, and dioleoyl phosphatidylethanolamine-polyethylene glycol.

[0014] In some specific embodiments, the biotinylated polyethylene glycol lipid is selected from distearoyl phosphatidylethanolamine-polyethylene glycol-biotin and / or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-biotin.

[0015] In some specific embodiments, the biotinylated polyethylene glycol lipid is selected from one or more of biotinylated polyethylene glycol-modified phospholipids, biotinylated polyethylene glycol-modified cholesterol, and biotinylated polyethylene glycol-modified fatty acids.

[0016] In some specific embodiments, the composite lipid nanoparticles are coated with nucleic acid molecules and / or drug molecules.

[0017] In some specific embodiments, the composite lipid nanoparticles are coated with nucleic acid molecules, and the molar ratio of the ionizable lipid nitrogen to the nucleic acid molecule phosphate is (4-6):1.

[0018] In some specific embodiments, the preparation of the recombinant antibody protein with a streptavidin tag includes: using seamless cloning technology to take the streptavidin coding gene and the antibody protein coding gene for construction to obtain a seamless cloning fragment; expressing the seamless cloning fragment to obtain the recombinant antibody protein with a streptavidin tag.

[0019] In some specific embodiments, the seamless cloning technology includes: taking an expression vector for restriction enzyme digestion to obtain a digestion product; taking the digestion product, the streptavidin coding gene, and the antibody protein coding gene for PCR amplification to obtain a PCR amplification product; transforming and screening the PCR amplification product to obtain a seamless cloning fragment.

[0020] In some specific embodiments, the expression vector is selected from the pCAGGS vector and / or the pGL3 vector.

[0021] In some specific embodiments, the expression vector is the pCAGGS vector, and the primer system for seamless cloning includes: SA-F1 with the nucleotide sequence shown in SEQ ID NO:1 and SA-R1 shown in SEQ ID NO:2; and / or, Ab-F1 with the nucleotide sequence shown in SEQ ID NO:3 and Ab-R1 shown in SEQ ID NO:4.

[0022] The preparation method of the composite lipid nanoparticles provided by the present invention includes: mixing the lipid composition and ethanol to obtain an organic phase solution; mixing the recombinant antibody protein with a streptavidin tag, an optional nucleic acid molecule, an optional drug and water to obtain an aqueous phase solution; mixing the organic phase solution and the aqueous phase solution to obtain the composite lipid nanoparticles.

[0023] In some specific embodiments, the concentration of the lipid composition in the organic phase solution is 50-75 wt%.

[0024] In some specific embodiments, the concentration of the recombinant antibody protein in the aqueous phase solution is 20-30 wt%, and the mixing volume ratio of the organic phase solution and the aqueous phase solution is 1:(1-5).

[0025] The RNA vaccine provided by the present invention includes the composite lipid nanoparticles described above.

[0026] The drug provided by the present invention includes the composite lipid nanoparticles described above.

[0027] The present invention provides the application of the composite lipid nanoparticles described above in nucleic acid and / or drug delivery.

[0028] Beneficial effects:

[0029] The present invention provides a composite lipid nanoparticle, which includes a lipid composition containing biotinylated polyethylene glycol lipid and a recombinant antibody protein with a streptavidin tag. First, by using the strong specific interaction between biotin and streptavidin, the connection between lipid nanoparticles and proteins can be achieved only by simple operations such as mixing. Second, by adopting the binding strategy of introducing biotin into lipid nanoparticles and streptavidin into antibody proteins, the recombinant antibody protein can bind to multiple biotinylated polyethylene glycol lipids, realizing its more stable connection with the antibody protein. In addition, introducing the streptavidin tag with the chemical nature of protein into the antibody protein will not interfere with the specific recognition ability of the antibody protein to the corresponding protein. This characteristic ensures that the composite lipid nanoparticles can accurately direct to target cells and / or tissues, thereby realizing the precise delivery of drugs and having better tissue delivery efficiency. Description of the drawings

[0030] Figure 1 It is the SEM image (scale bar is 200 nm) of the composite lipid nanoparticles provided in Example 1 of the present invention;

[0031] Figure 2 It is the experimental result image of the composite lipid nanoparticles transfected into macrophages provided in the test example of the present invention;

[0032] Figure 3 Experimental result graph of the experiment of transfecting mice with the composite lipid nanoparticles provided in the test example of the present invention. Detailed implementation manners

[0033] The composite lipid nanoparticles provided by the present invention specifically include a lipid composition and a recombinant antibody protein with a streptavidin tag. Among them, the lipid composition specifically includes an ionizable lipid, a helper lipid, cholesterol, a polyethylene glycol lipid, and a biotinylated polyethylene glycol lipid; and the mass ratio of the ionizable lipid, the helper lipid, cholesterol, the polyethylene glycol lipid, and the biotinylated polyethylene glycol lipid is (40-90):(0.1-20):(20-50):(0.5-10):(0.5-10), such as 40:0.1:20:0.5:0.5, 45:1:30:3:4, 75:5:37:6:10, 80:13:45:8:7, 90:20:50:10:5 or any value therebetween. The recombinant antibody protein with a streptavidin tag is to recombine and express the streptavidin-encoding gene and the antibody protein-encoding gene by means of biotechnology to achieve the splicing of the streptavidin protein and the antibody protein; at this time, compared with the existing chemical group modification of the antibody protein to endow it with reactivity with lipid nanoparticles, the specific recognition ability of the antibody protein provided by the present invention and the corresponding protein can better maintain the specific recognition ability of the antibody protein and the corresponding protein.

[0034] In the present invention, the ionizable lipid is a type of lipid having one or more hydrophilic groups and one or more hydrophobic groups. The ionizable property possessed both inside and outside cells endows it with the ability to freely cross the cell membrane. It is a type of substance commonly used in existing lipid nanoparticles and is not particularly limited. In some specific implementation manners, specific examples of the ionizable lipid include but are not limited to: trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleenyloxypropylammonium chloride, 3β-[N-(N’,N’-dimethylaminoethyl)carbamoyl] and 1-octyl nonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate, etc.

[0035] In the present invention, the helper lipid is a type of neutral lipid that plays a role in improving stability and in vivo circulation in the composite lipid nanoparticles. It is a type of substance commonly used in existing lipid nanoparticles and is not particularly limited. In some specific implementation manners, specific examples of the helper lipid include but are not limited to: dioleoyl phosphatidylethanolamine, stearoyl phosphatidylcholine, distearoyl phosphatidylcholine, and sterols, etc.

[0036] In the present invention, the polyethylene glycol lipid is a long-chain amphiphilic structure composed of a hydrophilic polyethylene glycol chain and a hydrophobic alkyl chain and / or dialkyl chain. Its addition can not only improve the particle size and particle stability of the composite lipid nanoparticles, but also affect aspects such as the nucleic acid encapsulation efficiency, half-life, transfection efficiency, and immune response of the composite lipid nanoparticles. It is a type of substance commonly used in existing lipid nanoparticles and is not particularly limited. In some specific embodiments, specific examples of the polyethylene glycol lipid include, but are not limited to, one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol, and dioleoyl phosphatidylethanolamine-polyethylene glycol.

[0037] In the present invention, the biotinylated polyethylene glycol lipid is a class of molecules in which biotin is connected to the polyethylene glycol lipid structure by chemical bonding. It can utilize the strong binding between biotin and streptavidin to achieve rapid connection with antibody proteins. It can be prepared by modifying the polyethylene glycol lipid with biotin, and the modification of the polyethylene glycol lipid with biotin is a commonly used modification method in the prior art and is not particularly limited. In some specific embodiments, specific examples of the biotinylated polyethylene glycol lipid include, but are not limited to, distearoyl phosphatidylethanolamine-polyethylene glycol-biotin and / or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-biotin.

[0038] In the present invention, based on the total mass of the lipid composition, the content of the ionizable lipid is preferably 40-60 wt%, such as 40 wt%, 43 wt%, 48 wt%, 50 wt%, 51.5 wt%, 53 wt%, 55 wt%, 58 wt%, 60 wt% or any value therebetween; the content of the helper lipid is preferably 5-20 wt%, such as 5 wt%, 5.8 wt%, 6 wt%, 6.5 wt%, 7 wt%, 9 wt%, 10 wt%, 12.5 wt%, 14 wt%, 18 wt%, 20 wt% or any value therebetween; the content of cholesterol is preferably 30-50 wt%, such as 30 wt%, 30.5 wt%, 32 wt%, 35 wt%, 38 wt%, 40 wt%, 43 wt%, 45 wt%, 50 wt% or any value therebetween; the content of the polyethylene glycol lipid is preferably 0.1-20 wt%, such as 0.1 wt%, 0.5 wt%, 0.8 wt%, 1 wt%, 1.3 wt%, 1.8 wt%, 2 wt%, 5 wt%, 9 wt%, 15 wt%, 18 wt%, 20 wt% or any value therebetween; the content of the biotinylated polyethylene glycol lipid is preferably 0.1-20 wt%, such as 0.1 wt%, 1 wt%, 3 wt%, 7 wt%, 10 wt%, 13 wt%, 18 wt%, 20 wt% or any value therebetween.

[0039] In the present invention, according to the application scenario, the composite lipid nanoparticles are preferably coated with nucleic acid molecules and / or drug molecules. In some specific embodiments, the composite lipid nanoparticles are used for delivering nucleic acid molecules such as DNA, mRNA, siRNA, circRNA, etc., and the molar ratio of the ionizable lipid nitrogen to the nucleic acid molecule phosphate is preferably (4-6):1. Such as 4:1, 4.05:1, 4.3:1, 4.8:1, 5:1, 5.5:1, 6:1 or any value therebetween.

[0040] In the present invention, the preparation of the recombinant antibody protein with a streptavidin tag preferably includes: using seamless cloning technology to construct the streptavidin coding gene and the antibody protein coding gene to obtain a seamless cloning fragment; expressing the seamless cloning fragment to obtain the recombinant antibody protein with a streptavidin tag.

[0041] In the present invention, the seamless cloning technology is a biotechnology for assembling a plasmid vector linearized by any method with one or more DNA fragments and constructing multi-site mutations, which has the advantages of being fast, simple, efficient, multi-fragment assembly and directional cloning.

[0042] In the present invention, the seamless cloning technology preferably includes: subjecting an expression vector to restriction digestion to obtain a restriction digestion product; subjecting the restriction digestion product, streptavidin coding gene and antibody protein coding gene to PCR amplification to obtain a PCR amplification product; and subjecting the PCR amplification product to transformation and screening to obtain a seamless cloning fragment.

[0043] In some specific embodiments, the enzyme system for the restriction digestion reaction includes but is not limited to: restriction endonuclease and / or T5 exonuclease. The conditions for the restriction digestion reaction include that the temperature is preferably 30-35 °C, such as 30 °C, 30.5 °C, 31 °C, 32 °C, 33 °C, 35 °C or any value therebetween; and the time is preferably 30-60 min, such as 30 min, 35 min, 38 min, 45 min, 50 min, 55 min, 60 min or any value therebetween.

[0044] In some specific embodiments, the expression vector is a type of substance commonly used in seamless cloning technology, and no special limitation is imposed thereon. Specific examples include but are not limited to: pCAGGS vector and / or pGL3 vector. In some more preferred embodiments, the expression vector is preferably the pCAGGS vector, and the primer system for seamless cloning preferably includes: SA-F1 with the nucleotide sequence shown in SEQ ID NO:1 and SA-F1 shown in SEQ ID NO:2; and / or, Ab-F1 with the nucleotide sequence shown in SEQ ID NO:3 and Ab-F1 shown in SEQ ID NO:4. At this time, the seamless cloning fragment obtained by PCR amplification, transformation and screening through this primer system has a better specific binding ability with related proteins when the recombinant antibody protein with streptavidin tag expressed thereby.

[0045] The preparation method of the above-mentioned composite lipid nanoparticles provided by the present invention specifically includes: mixing the lipid composition and ethanol to obtain an organic phase solution; mixing the recombinant antibody protein with streptavidin tag, an optional nucleic acid molecule, an optional drug and water to obtain an aqueous phase solution; and mixing the organic phase solution and the aqueous phase solution to obtain the composite lipid nanoparticles.

[0046] In the present invention, the input mass ratio of the lipid composition to ethanol is preferably such that the concentration of the lipid composition in the organic phase solution is 50-75 wt%, such as 50 wt%, 55 wt%, 58 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt% or any value therebetween. At this time, the concentration of the lipid composition in the organic phase solution is appropriate, which is conducive to the formation of composite lipid nanoparticles with good morphology and high particle size uniformity.

[0047] In the present invention, during the mixing process of the organic phase solution and the aqueous phase solution, composite lipid nanoparticles gradually precipitate and solidify, while achieving the insertion with the recombinant antibody protein with streptavidin tags and encapsulating nucleic acid molecules and / or drugs; the method for mixing the organic phase solution and the aqueous phase solution is a method commonly used in the preparation of existing composite lipid nanoparticles and is not particularly limited. Specific examples thereof include, but are not limited to, one or more of microfluidic mixing technology, thin film hydration method, extrusion method, injection method, and homogenization method. In some specific embodiments, the concentration of the recombinant antibody protein in the aqueous phase solution is 20-30 wt%, such as 20 wt%, 21 wt%, 23 wt%, 25 wt%, 27.5 wt%, 28 wt%, 30 wt% or any value therebetween, and the mixing volume ratio of the organic phase solution to the aqueous phase solution is preferably 1:(1-5), such as 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5 or any value therebetween. At this time, the ratios of the recombinant protein to the lipid composition, and the organic phase solution to the aqueous phase solution are appropriate, such that a relatively ideal distribution and three-dimensional structure of the recombinant antibody protein are formed on the composite lipid nanoparticles, thereby achieving a better drug delivery effect.

[0048] The RNA vaccine provided by the present invention specifically includes the above-mentioned composite lipid nanoparticles. Through the encapsulation of RNA by the composite lipid nanoparticles, RNA can be safely and stably delivered to target cells and / or tissues, thereby achieving a good immune response effect.

[0049] The drug provided by the present invention specifically includes the above-mentioned composite lipid nanoparticles. Through the encapsulation of drug molecules by the composite lipid nanoparticles, the drug molecules can be safely and stably delivered to target cells and / or tissues, thereby achieving a good therapeutic effect.

[0050] The present invention also provides the application of the above composite lipid nanoparticles in the field of nucleic acid and / or drug delivery.

[0051] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. For those not specifying specific techniques or conditions in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0052] The reagents and their sources involved in the following examples and comparative examples are specifically as follows:

[0053] LB solid medium with sodium ampicillin (Shanghai Sangon, product number B530112);

[0054] Ionizable lipid (A.V.T., product number: O02010);

[0055] Helper lipid (A.V.T., product number: DSPC);

[0056] Cholesterol (Sigma, product number: C8667);

[0057] Polyethylene glycol lipid (A.V.T., product number: O02005);

[0058] Biotinylated polyethylene glycol lipid (Xi'an Ruixi Biotechnology, product number: R-1304-2k); DEME medium (Gbico, product number: C11995500BT);

[0059] Streptavidin (Yeasen, product number: 35100ES03).

[0060] The nucleotide sequences involved in the following examples and comparative examples are shown in Table 1:

[0061] Table 1. Nucleotide sequence list

[0062]

[0063]

[0064] Example 1

[0065] This example is used to illustrate the preparation of a composite lipid nanoparticle, which specifically includes:

[0066] 1. Construction of a recombinant antibody protein with a streptavidin tag: (1) Obtain the streptavidin-encoding gene and antibody protein-encoding gene with nucleotide sequences shown in SEQ ID NO:5 and 6 respectively by gene synthesis. Use a restriction enzyme to digest the PCAGGS vector at 37°C for 30 minutes to obtain a digestion product;

[0067] (2) Use SA-F1 with the nucleotide sequence shown in SEQ ID NO:1, SA-R1 shown in SEQ ID NO:2, Ab-F1 with the nucleotide sequence shown in SEQ ID NO:3, and Ab-R1 shown in SEQ ID NO:4 to perform PCR amplification on the streptavidin-encoding gene and antibody protein-encoding gene to obtain a PCR amplification product;

[0068] The PCR reaction system includes: 1 μL of streptavidin encoding gene, or 1 μL of antibody protein encoding gene, 1 μL of SA-F1, 1 μL of SA-R1, or 1 μL of Ab-F1, 1 μL of Ab-R1, 10 μL of 5×fastpfu buffer, 4 μL of dNTPs (2.5 mM), 1.5 μL of Fast pfu, and ddH2O is added to a total volume of 50 μL; the reaction conditions include: pre-denaturation at 94°C for 60 s, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 5 min, and 30 cycles.

[0069] (3) Take 5 μL of the PCR amplification product and transfect Escherichia coli DH5ɑ competent cells (cell density of 70%) for 24 hours, then spread it on LB solid medium containing ampicillin sodium, pick a single colony with good growth status and send it for sequencing, identify and screen, and obtain seamless clone fragments;

[0070] (3) After transfecting 293f cells (cell density 70%) with the seamless cloned fragment for 96 hours, the culture medium was collected and purified by gel electrophoresis to obtain a recombinant antibody protein with a streptavidin tag.

[0071] 2. Preparation of composite lipid nanoparticles by ethanol injection method: (1) 275 parts by weight of ionizable lipid, 70 parts by weight of auxiliary lipid, 120 parts by weight of cholesterol, 7.5 parts by weight of polyethylene glycol lipid, 9 parts by weight of biotinylated polyethylene glycol lipid and 100 parts by weight of ethanol were mixed thoroughly to obtain an organic phase solution;

[0072] (2) 100 parts by weight of a recombinant antibody protein with a streptavidin tag, 20 parts by weight of luciferase-CY5 mRNA, and 300 parts by weight of water were mixed to obtain an aqueous solution;

[0073] (3) The aqueous phase solution was quickly added to the organic phase solution at a volume ratio of 3:1, mixed evenly and incubated for 30 minutes to obtain a suspension containing composite lipid nanoparticles. The suspension was ultrafiltered and centrifuged to obtain composite lipid nanoparticles.

[0074] Figure 1 The following is a SEM image of the composite lipid nanoparticles prepared by the present invention. Figure 1 It can be seen that the composite lipid nanoparticles have a relatively regular spherical structure, and a certain amount of recombinant antibody protein with a streptavidin tag is successfully connected to its surface.

[0075] Example 2

[0076] This example prepares composite lipid nanoparticles according to the method provided in Example 1. The difference is that in step 2(1), the addition amount of the ionizable lipid is 40.75 parts by mass, and the addition amount of the biotinylated polyethylene glycol lipid is 10 parts by mass. Other conditions are the same, and composite lipid nanoparticles are obtained.

[0077] Example 3

[0078] This example prepares composite lipid nanoparticles according to the method provided in Example 1. The difference is that in step 2(1), the addition amount of the ionizable lipid is 65 parts by mass, the addition amount of the co-lipid is 8 parts by mass, and the addition amount of cholesterol is 25.5 parts by mass. Other conditions are the same, and composite lipid nanoparticles are obtained.

[0079] Example 4

[0080] This example prepares composite lipid nanoparticles according to the method provided in Example 1. The difference is that in step 2(3), the volume ratio of the aqueous phase solution to the organic phase solution is 4:1. Other conditions are the same, and composite lipid nanoparticles are obtained.

[0081] Comparative Example 1

[0082] This comparative example prepares composite lipid nanoparticles according to the method provided in Example 1. The difference is that in step 2(1), the biotinylated antibody protein is used to replace the recombinant antibody protein with a streptavidin tag in equal parts by mass, and 15 parts by mass of streptavidin is added. Other conditions are the same, and composite lipid nanoparticles are obtained.

[0083] Among them, the preparation of the biotinylated antibody protein includes: obtaining the antibody protein coding gene with the nucleotide sequence shown in SEQ ID NO:6 by gene synthesis, performing enzymatic digestion on the PCAGGS vector at 37°C for 30 min using a restriction endonuclease to obtain the enzymatic digestion product, and expressing the antibody protein with reference to the method of "construction of the recombinant antibody protein with a streptavidin tag" provided in Example 1;

[0084] Then transfer the antibody protein to 1× modified buffer (containing 100 mM phosphoric acid and 150 mM sodium chloride, pH 7.2 - 7.4) to prepare an antibody solution with a protein concentration of 5 mg / L; take 0.8 L of biotin solution (dimethylformamide solution containing 20 g / L biotin); add 0.8 L of biotin solution to 1 L of the antibody solution, stir and incubate at room temperature for 2 h, then centrifuge and filter to obtain a solution containing the biotinylated antibody protein.

[0085] Comparative Example 2

[0086] This comparative example prepared composite lipid nanoparticles according to the method provided in Example 1. The difference is that in (1) of Step 2, polyethylene glycol lipid in equal mass parts was used to replace biotinylated polyethylene glycol lipid, and other conditions were the same, obtaining composite lipid nanoparticles.

[0087] Comparative Example 3

[0088] This comparative example prepared composite lipid nanoparticles according to the method provided in Example 1. The difference is that in (2) of Step 2, antibody protein in equal mass parts was used to replace the recombinant antibody protein with streptavidin tag, and other conditions were the same, obtaining composite lipid nanoparticles.

[0089] Among them, the preparation of the antibody protein referred to Comparative Example 1.

[0090] Comparative Example 4

[0091] This comparative example prepared composite lipid nanoparticles according to the method provided in Example 1. The difference is that in (2) of Step 2, water in equal mass parts was used to replace the recombinant antibody protein with streptavidin tag, and other conditions were the same, obtaining composite lipid nanoparticles.

[0092] Test Example

[0093] This test example was used to illustrate the delivery efficiency and immunogenicity of the composite lipid nanoparticles provided in Examples 1-4 and Comparative Examples 1-3, specifically including:

[0094] 1. Transfection of macrophages with composite lipid nanoparticles: (1) Macrophages were taken and added to DEME medium, and cultured in a CO2 incubator at 5% CO2, saturated humidity and 37 °C until the cell density was about 70%;

[0095] (2) 0.2 μg of composite lipid nanoparticles was taken and added to the medium, and incubated in a CO2 incubator at 5% CO2, saturated humidity and 37 °C for 4 h, and the CY5 fluorescence signal was detected. The test results are shown in Table 2 and Figure 2 as follows.

[0096] Table 2. Ability of composite lipid nanoparticles to transfect macrophages

[0097] Group CY5 Fluorescence Signal Example 1 178500 Example 2 182000 Example 3 184000 Example 4 176000 Comparative Example 1 112000 Comparative Example 2 115000 Comparative Example 3 117000 Comparative Example 4 118000

[0098] From the test results shown in Table 2 and Figure 2 as follows, it can be seen that compared with Comparative Examples 1-4, the CY5 fluorescence signal shown by the macrophages transfected with the composite lipid nanoparticles provided in Examples 1-4 of the present invention is at least 1.4 times higher than that of Comparative Examples 1-4, indicating that the composite lipid nanoparticles provided in Examples 1-4 of the present invention can better recognize and bind to macrophages and achieve an ideal transfection effect.

[0099] 2. Transfecting mice with composite lipid nanoparticles: BALB / c Nude nude mice (male, 6 - 8 weeks old, under SPF - level breeding conditions) were used as experimental animals. A needle was inserted into the right abdominal cavity of the nude mice, about 5 mm deep into the abdominal cavity. 5 μg of the composite lipid nanoparticles provided in Example 1 and Comparative Example 3 were respectively injected intraperitoneally. After the injection, the needle was withdrawn while rotating, and the needle hole was pressed with a cotton swab until no liquid flowed out to complete the intraperitoneal injection. On the 1st day after the injection, the mice were dissected, and fluorescence imaging was performed on their heart, liver, spleen, lung, and kidney (organs). The results are as Figure 3 shown.

[0100] As Figure 3 can be seen, under the same mouse breeding and treatment conditions, compared with Comparative Example 3, when the composite lipid nanoparticles provided in Example 1 of the present invention were used to transfect mice, stronger fluorescence signals were presented in the heart, liver, spleen, lung, and kidney (organs), that is, the luciferase - CY5 mRNA encapsulated in the composite lipid nanoparticles had better delivery efficiency for mice.

[0101] Although the embodiments of the present invention have been shown and described above, it can be understood that the above - mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above - mentioned embodiments within the scope of the present invention without departing from the principle and purpose of the present invention.

Claims

1. A composite lipid nanoparticle, characterized in that, The composite lipid nanoparticles comprise a lipid composition and a recombinant antibody protein with a streptavidin tag; The lipid composition comprises an ionizable lipid, a co-lipid, cholesterol, a polyethylene glycol lipid, and a biotinylated polyethylene glycol lipid, and the mass ratio of the ionizable lipid, co-lipid, cholesterol, polyethylene glycol lipid, and biotinylated polyethylene glycol lipid is (40-90):(0.1-20):(20-50):(0.5-10):(0.5-10); The recombinant antibody protein with a streptavidin tag is an amino acid fragment translated from the nucleotide fragment shown in SEQ ID NO:6; The ionizable lipid is selected from one or more of trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleenyloxypropylammonium chloride, 3β-[N-(N’,N’-dimethylaminoethyl)carbamoyl], and 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate; The co-lipid is selected from one or more of dioleoyl phosphatidylethanolamine, stearoyl phosphatidylcholine, distearoyl phosphatidylcholine, and sterols; The polyethylene glycol lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol, and dioleoyl phosphatidylethanolamine-polyethylene glycol; The biotinylated polyethylene glycol lipid is selected from one or more of distearoyl phosphatidylethanolamine-polyethylene glycol-biotin and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-biotin.

2. The composite lipid nanoparticles according to claim 1, wherein Based on the weight of the lipid composition, the content of the ionizable lipid is 40-60 wt%, the content of the co-lipid is 5-20 wt%, the content of cholesterol is 30-50 wt%, the content of the polyethylene glycol lipid is 0.1-20 wt%, and the content of the biotinylated polyethylene glycol lipid is 0.1-20 wt%.

3. The composite lipid nanoparticles according to claim 1, wherein The composite lipid nanoparticles are coated with nucleic acid molecules and / or drug molecules.

4. The composite lipid nanoparticles according to claim 1, characterized in that, The composite lipid nanoparticles are coated with nucleic acid molecules, and the molar ratio of the ionizable lipid nitrogen to the nucleic acid molecule phosphate is (4-6):

1.

5. The composite lipid nanoparticles according to claim 1, characterized in that, The preparation of the recombinant antibody protein with a streptavidin tag includes: using seamless cloning technology to construct the streptavidin coding gene and the antibody protein coding gene to obtain a seamless cloning fragment; expressing the seamless cloning fragment to obtain the recombinant antibody protein with a streptavidin tag.

6. The composite lipid nanoparticles according to claim 5, characterized in that, The seamless cloning technology includes: subjecting an expression vector to restriction enzyme digestion to obtain a digestion product; performing PCR amplification on the digestion product, the streptavidin coding gene, and the antibody protein coding gene to obtain a PCR amplification product; transforming and screening the PCR amplification product to obtain a seamless cloning fragment.

7. The composite lipid nanoparticles according to claim 6, characterized in that, The expression vector is selected from pCAGGS vector and / or pGL3 vector.

8. The composite lipid nanoparticles according to claim 6, characterized in that, The expression vector is the pCAGGS vector, and the primer system for seamless cloning includes: SA-F1 with a nucleotide sequence as shown in SEQ ID NO:1 and SA-R1 with a nucleotide sequence as shown in SEQ ID NO:2; and / or, Ab-F1 with a nucleotide sequence as shown in SEQ ID NO:3 and Ab-R1 with a nucleotide sequence as shown in SEQ ID NO:

4.

9. The preparation method of the composite lipid nanoparticles according to any one of claims 1 to 8, characterized in that, The preparation method includes: mixing the lipid composition and ethanol to obtain an organic phase solution; mixing the recombinant antibody protein with a streptavidin tag, an optional nucleic acid molecule, an optional drug, and water to obtain an aqueous phase solution; mixing the organic phase solution and the aqueous phase solution to obtain the composite lipid nanoparticles.

10. The preparation method of the composite lipid nanoparticles according to claim 9, characterized in that, The concentration of the lipid composition in the organic phase solution is 50-75 wt%.

11. The preparation method of the composite lipid nanoparticles according to claim 9, characterized in that, The concentration of the recombinant antibody protein in the aqueous phase solution is 20-30 wt%, and the mixing volume ratio of the organic phase solution to the aqueous phase solution is 1:(1-5).

12. An RNA vaccine, characterized in that, The RNA vaccine includes the composite lipid nanoparticles according to any one of claims 1-8.

13. A drug, characterized in that, The drug includes the composite lipid nanoparticles according to any one of claims 1-8.

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