Albumin site-specific modified lipid nanoparticles as well as preparation and application thereof

Abstract

The invention discloses an albumin site-directed modified lipid nanoparticle as well as preparation and application thereof, a nucleic acid-loaded lipid nanoparticle is prepared from a synthesized functionalized low molecular weight polyethylene glycol (PEG) lipid compound-maleimide derivative and a commonly used nucleic acid delivery lipid composition material, and the nucleic acid-loaded lipid nanoparticle can be used for preparing the albumin site-directed modified lipid nanoparticle. And coupling maleimide on the surface of the lipid nanoparticle with sulfydryl of albumin to prepare the lipid nanoparticle of which the surface is subjected to site-specific modification by the albumin. According to the invention, the immunogenicity of the existing mRNA-loaded lipid nanoparticles, which is generated by covering PEG on the outer layer of the nanoparticles, is reduced, the targeting property of related immune organs of the nanoparticles is improved, mRNA is effectively delivered to lymph nodes to induce an anti-tumor effect, and a foundation is laid for further development of the mRNA into a novel vaccine capable of activating an immune system to generate an anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a nanocarrier and a nanodrug system, and specifically to an albumin site-modified lipid nanoparticle, a drug-loaded lipid nanoparticle, a preparation method thereof, and an application thereof in mRNA delivery and anti-tumor. Background Art

[0002] Messenger RNA (mRNA) is transcribed from a DNA template, carries the genetic information encoding the target protein, and can be translated into functional protein under the action of ribosomes. This can be used to deliver exogenous mRNA into cells to obtain the required functional protein, thereby playing a role in treating or preventing diseases.

[0003] In recent decades, with the continuous development of vaccine technology, mRNA vaccines have made breakthrough progress. Currently, tumor vaccines and other immunotherapies are considered to be very promising methods for treating malignant tumors. The design of tumor vaccines can focus on antigens specifically expressed by tumor cells, such as growth factors, or on new antigens produced by tumor cell mutations. Cancer mRNA vaccines are mainly used for treatment, and their goal is to stimulate the body's immune response, especially T cell-mediated immune response, to eliminate or reduce the number of tumor cells.

[0004] Since mRNA is unstable and immunogenic under physiological conditions, and has the same negative charge as the cell membrane (making it difficult to enter the cell to exert its effect), the research focus of mRNA vaccines is mainly on two areas: the delivery system and the selection of tumor antigens. Viral vectors were once an important tool for achieving efficient expression of exogenous genes due to their efficient transfection and expression capabilities. However, due to the immune response that viral vectors may induce, the lack of specificity, and the limitation of mRNA length, they have gradually been replaced by non-viral vectors represented by cationic lipid nanoparticles, and have become the mainstream choice for gene delivery research.

[0005] The delivery systems of the two mRNA vaccines currently on the market (from Pfizer / BioNTech and Moderna) are both lipid nanoparticles (LNP). Both have a particle size of 80~100nm, and their composition is also very similar, both containing the following 4 components: 1) ionizable cationic lipids; 2) PEGylated lipids; 3) cholesterol; 4) phospholipid derivatives (such as distearoylphosphatidylcholine, DSPC). At present, a lot of results have been achieved in improving lipid composition and optimizing for specific cells or tissues. For example: CN116350757A demonstrated the enhancement of CAR-T cell generation in vivo by binding to CD5 antibodies; while CN117015374A prepared a new type of LNP by adjusting the lipid composition to improve delivery efficiency. In addition, CN115925975A constructed a (PA)2 peptide nano-micelle carrying a CD133-CAR plasmid, and modified it with a targeting group of macrophage-specific target CD206, citric anhydride-modified dextran, which can perform CAR editing on macrophages in vivo and in vitro. Despite this, the performance of LNPs for delivering mRNA still has a lot of room for improvement in terms of improving biocompatibility and transfection efficiency, including: reducing the immunogenicity of the existing vaccine (mRNA-carrying lipid nanoparticles) covered with PEG on the outer layer to the body, and improving the targeting of the vaccine, delivering mRNA to relevant immune organs (lymph nodes), thereby activating the immune system to produce anti-tumor effects. Summary of the invention

[0006] In view of the fact that existing nucleic acid (such as mRNA) lipid nanoparticles have insufficient targeting to lymph nodes and the immunogenicity caused by the PEG chains on their surface, the present invention provides a functionalized low molecular weight PEG-maleimide lipid compound, which is used together with commonly used nucleic acid delivery lipid composition materials (other conventional lipid nanoparticle raw materials) to prepare nucleic acid-loaded lipid nanoparticles, and the maleimide on the surface of the nanoparticles is chemically coupled with the only thiol group of albumin through Michael addition reaction, thereby obtaining nanoparticles with albumin site-specific modification on the surface of the lipid nanoparticles.

[0007] The present invention adopts the following technical scheme.

[0008] A lipid nanoparticle with albumin site-specific modification comprises the lipid nanoparticle and albumin on its surface.

[0009] A drug-loaded lipid nanoparticle modified with albumin at a fixed point, comprising the above-mentioned lipid nanoparticle modified with albumin at a fixed point and a drug.

[0010] In the present invention, in the lipid nanoparticles modified with albumin at a specific site, the raw materials for preparing the lipid nanoparticles include lipid compounds and existing lipid composition materials.

[0011] The invention discloses a lipid compound, which contains a hydrophobic segment, a hydrophilic segment and a group that can be coupled with a protein; the hydrophobic segment and the group that can be coupled with a protein are respectively located at the ends of the lipid compound.

[0012] Furthermore, in the lipid compound, the hydrophobic segment includes an alkyl segment; the hydrophilic segment includes an ethylene glycol segment, a hydrophilic functional polypeptide segment or a PEG-like segment; and the group that can be coupled to a protein includes a maleimide group. Specifically, the maleimide is at the hydrophilic tail, and the alkyl chain is the entirety, serving as the hydrophobic tail.

[0013] The lipid compound of the present invention is used together with conventional raw materials for preparing lipid nanoparticles, or the lipid compound of the present invention is used together with conventional raw materials and drugs for preparing lipid nanoparticles to prepare drug-loaded lipid nanoparticles; the nanoparticles have maleimide groups and can be coupled with proteins, thereby obtaining nanoparticles with albumin site-specific modification on the surface of the lipid nanoparticles.

[0014] Specifically, the chemical structural formula of the lipid compound is as follows: ; Among them, R1 and R2 are -C(=O)OR respectively, wherein R is a substituted or unsubstituted alkyl group; X is the residue of a compound containing amino and carboxyl groups after substitution; Y includes an ethylene glycol segment; n is 1 to 5, preferably 1 to 3; and Mal is a maleimide group.

[0015] In the present invention, the substituents in the substituted alkyl group include alkyl, halogen, nitro and the like.

[0016] In the present invention, the alkyl group includes a straight-chain alkyl group, a branched-chain alkyl group, and a cycloalkyl group.

[0017] In the present invention, the number of carbon atoms in the alkyl group is 1 to 30; preferably, the number of carbon atoms in the alkyl group is 3 to 27; more preferably, the number of carbon atoms in the alkyl group is 6 to 25; as an example, the alkyl group includes unsubstituted C8-C 24 Straight chain alkyl, unsubstituted C6-C 18 branched alkyl, straight chain alkyl substituted by C3-C8 cycloalkyl, or unsubstituted C8-C 24 Cycloalkyl.

[0018] In the present invention, X is the residue of a compound (amino acid) containing an amino group and a carboxyl group after substitution, connected to the hydrophobic segment via an ester bond, and connected to Y via an amide bond. Preferably, X is an amino acid residue.

[0019] In the present invention, amino acids include glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, sarcosine and histidine, selenocysteine ​​and pyrrolysine, etc.

[0020] In the present invention, amino acid residue is a conventional expression, generally referring to the group after the hydroxyl group of the carboxyl group and / or the hydrogen on the amine group of the amino acid are removed, that is, the residue part after being substituted.

[0021] In the present invention, Y is a chain segment containing ethylene glycol, a hydrophilic functional polypeptide chain (such as a protein polypeptide composed of multiple amino acid residues) or a PEG-like chain, such as a polyamino acid chain, a polyacrylamide chain, and the like.

[0022] Preferably, Y contains an ethylene glycol segment and also contains an amide group and / or an amino acid residue. Y is a straight chain or a straight chain structure, and preferably, Y is a straight chain containing a carbonyl group.

[0023] In the present invention, the ethylene glycol segment is a segment having ethylene glycol repeating units; specifically, in the ethylene glycol segment contained in Y, the number of ethylene glycol repeating units is 1 to 25, preferably, the number of ethylene glycol repeating units is 2 to 20, more preferably, the number of ethylene glycol repeating units is 3 to 18, and further preferably, the number of ethylene glycol repeating units is 4 to 16.

[0024] In the present invention, Y is connected to maleimide through an amide bond. Preferably, there is a substituted or unsubstituted alkyl group between the amide bond and the maleimide group. Preferably, the alkyl group contains 1 to 20 carbon atoms, more preferably, contains 2 to 15 carbon atoms, and even more preferably, contains 3 to 10 carbon atoms, such as 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms or any value within the range.

[0025] In the present invention, the raw materials for preparing lipid nanoparticles include other raw materials in addition to the above-mentioned lipid compounds. The other raw materials are other conventional lipid nanoparticle raw materials, which are existing technologies, such as: ionizable cationic lipids, cholesterol, phospholipid derivatives, polyethylene glycol lipids, etc.; as an example, the other raw materials are ionizable cationic lipids, cholesterol, phospholipid derivatives and polyethylene glycol lipids.

[0026] The present invention discloses a method for preparing the lipid nanoparticles modified with albumin at a fixed point, wherein lipid nanoparticle raw materials are used as raw materials to prepare lipid nanoparticles, and then albumin is modified on the lipid nanoparticles to obtain lipid nanoparticles modified with albumin at a fixed point; specifically, lipid nanoparticle raw materials including the lipid compound are used as raw materials to prepare lipid nanoparticles, and then albumin is modified on the lipid nanoparticles to obtain lipid nanoparticles modified with albumin at a fixed point. In addition to the lipid compound, the lipid nanoparticle raw materials including the lipid compound also include other conventional lipid nanoparticle raw materials, such as ionizable cationic lipids, cholesterol, phospholipid derivatives, polyethylene glycol lipids, etc.

[0027] The present invention discloses a method for preparing drug-loaded lipid nanoparticles modified with albumin at a fixed point, wherein the drug-loaded lipid nanoparticles are prepared with lipid nanoparticle raw materials and drugs as raw materials, and then albumin is modified on the drug-loaded lipid nanoparticles to obtain the drug-loaded lipid nanoparticles modified with albumin at a fixed point; specifically, lipid nanoparticle raw materials including the lipid compound and drugs are prepared with lipid nanoparticles, and then albumin is modified on the lipid nanoparticles to obtain the lipid nanoparticles modified with albumin at a fixed point. The lipid nanoparticle raw materials including the lipid compound include other conventional lipid nanoparticle raw materials, such as ionizable cationic lipids, cholesterol, phospholipid derivatives, polyethylene glycol lipids, etc., in addition to the above-mentioned lipid compounds.

[0028] As an example, the preparation method of drug-loaded lipid nanoparticles includes the following steps: the aqueous phase containing nucleic acid and the lipid organic phase are mixed by physical method or microfluidic method to prepare nucleic acid-loaded lipid nanoparticles. The specific preparation of the aqueous phase and the organic phase is conventional technology, and water and alcohol can be used as solvents respectively.

[0029] In the present invention, the molar ratio of the lipid compound to other raw materials is 1% to 35%, preferably 1.5% to 20%, more preferably 1.5% to 15%, further preferably 2% to 10%, and further preferably 3% to 6%.

[0030] In the present invention, the drug includes nucleic acid, such as one or more of mRNA, circular RNA, siRNA, microRNA, and antisense nucleic acid.

[0031] In the present invention, the albumin contains a thiol group, such as human serum albumin and bovine serum albumin.

[0032] In the present invention, lipid nanoparticles are mixed and incubated with albumin to obtain lipid nanoparticles modified with albumin at fixed points; and drug-loaded lipid nanoparticles are mixed and incubated with albumin to obtain drug-loaded lipid nanoparticles modified with albumin at fixed points.

[0033] In the present invention, the incubation temperature is 0°C to room temperature; preferably, the lipid nanoparticles are mixed with albumin and incubated at room temperature, and then allowed to stand at 0°C to 5°C to obtain lipid nanoparticles with site-specific modification of albumin; the drug-loaded lipid nanoparticles are mixed with albumin and incubated at room temperature, and then allowed to stand at 0°C to 5°C to obtain drug-loaded lipid nanoparticles with site-specific modification of albumin.

[0034] The drug-loaded liposome nanoparticles of the present invention are a nucleic acid (mRNA) delivery system for albumin site-specific modification targeting lymph nodes. At low pH values, it is beneficial for mRNA endosomal escape to exert translation function. At physiological pH values, it is neutral and significantly reduces potential safety toxicity. At the same time, it can reduce antibody binding to serum proteins and clearance by phagocytes, prolong systemic circulation time, and is not prone to the risk of allergic reactions. It has the effect of stabilizing and promoting membrane fusion. The nanoparticles of the present invention have natural lymph node targeting ability, can significantly enhance the uptake capacity of antigen presenting cells, thereby efficiently activating the body's immune response. Moreover, LNP is easy to design and prepare, and can produce vaccines that are both effective and safe, so it has good application prospects.

[0035] The invention discloses the use of the lipid compound, albumin site-modified lipid nanoparticles or albumin site-modified drug-loaded lipid nanoparticles in the preparation of biological products or drugs.

[0036] The invention discloses the application of the lipid compound, albumin site-specific modified lipid nanoparticles or albumin site-specific modified drug-loaded lipid nanoparticles in the preparation of anti-tumor drug reagents.

[0037] A biological product, the active ingredient of which includes the above-mentioned albumin site-modified drug-loaded lipid nanoparticles.

[0038] In the present invention, the biological product is a vaccine, preferably a nucleic acid vaccine, such as an mRNA vaccine; it includes infectious disease vaccines or tumor vaccines: influenza vaccines, AIDS vaccines, viral pneumonia vaccines, tuberculosis vaccines, respiratory syncytial virus vaccines, enterovirus vaccines, intestinal mucosa-associated tumor vaccines or tumor vaccines, etc.

[0039] Preferably, the biological product is used to prevent and / or treat infectious diseases or tumors; tumors include solid tumors or blood tumors, such as melanoma, lung cancer, colon cancer and other types of tumors.

[0040] Furthermore, the present invention discloses an anti-tumor drug, the active ingredient of which includes the above-mentioned albumin site-specifically modified drug-loaded lipid nanoparticles.

[0041] In the present invention, the biological product or drug can be applied by injection, oral administration, external application, etc.

[0042] The present invention uses the above-mentioned albumin site-specific modified drug-loaded lipid nanoparticles as the only active ingredient of the anti-tumor drug, and the above-mentioned albumin site-specific modified drug-loaded lipid nanoparticles can also be used in combination with other anti-tumor drugs for treatment.

[0043] The present invention prepares lipid nanoparticles capable of carrying nucleic acid by combining lipid compounds (functionalized low molecular weight PEG-maleimide derivatives) with commonly used nucleic acid delivery lipid raw materials; nucleic acid is added to the lipid nanoparticle preparation raw materials to prepare nucleic acid-carrying lipid nanoparticles; and maleimide on the surface of the nanoparticles is chemically coupled with the only thiol group of albumin through Michael addition reaction, thereby obtaining nucleic acid-carrying lipid nanoparticles mRNA-LNP with albumin site-specifically coupled on the surface.

[0044] The present invention provides a lipid nanoparticle modified with albumin, a preparation method thereof, and application in mRNA delivery and anti-tumor. The advantages of the above-mentioned albumin-site-modified nanoparticles are as follows: 1) The maleimide on the surface of the nanoparticle is chemically coupled with the only thiol group of albumin, so that the albumin site-modified nanoparticle surface is realized, causing steric hindrance to the anti-PEG chain antibody, and reducing the immunogenicity of the nanoparticles to the body; 2) The lymph node targeting of the albumin site-coupled on the surface can be used to improve the lymph node enrichment of the nanoparticles and better play a role. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Lipid nanoparticles with site-specific modification of albumin.

[0046] Figure 2 Schematic diagram of the preparation of intermediate DMG-G.

[0047] Figure 3 Schematic diagram of the preparation of the intermediate Mal-PEG4-OH.

[0048] Figure 4 For the intermediates Mal-PEG8-OH, Mal-PEG 12 -OH, Mal-PEG 16 Schematic diagram of the preparation of -OH.

[0049] Figure 5 Schematic diagram of the preparation of lipid compounds.

[0050] Figure 6 Fluorescence distribution of nanoparticle-delivered drugs in lymph nodes: A. Lymph nodes photographed by a small animal imager; B. Histogram of fluorescence values.

[0051] Figure 7Figure 3 is the tumor growth curve of B16-OVA tumor-bearing mice treated with nanoparticles. The data were processed according to conventional statistical methods. ns indicates no statistical difference, * indicates P<0.05, and ** indicates P<0.01. DETAILED DESCRIPTION

[0052] The present invention belongs to the field of biomedicine technology, and specifically relates to a functionalized low molecular weight PEG-maleimide lipid compound, which is prepared together with a commonly used nucleic acid delivery lipid composition material to obtain nucleic acid-loaded lipid nanoparticles, and the maleimide on the surface of the nanoparticles is chemically coupled with the only thiol group of albumin through a Michael addition reaction, thereby obtaining nanoparticles with albumin site-specific modification on the surface of the lipid nanoparticles.

[0053] In view of the fact that existing mRNA-carrying lipid nanoparticles have insufficient targeting to lymph nodes and the immunogenicity caused by the PEG chains on their surface, the present invention provides a functionalized low molecular weight PEG-maleimide derivative and a preparation method thereof, which is used together with a commonly used nucleic acid delivery lipid composition material to prepare nucleic acid-carrying lipid nanoparticles, and the maleimide on the surface of the nanoparticles is chemically coupled with the only thiol group of albumin through a Michael addition reaction, thereby obtaining nanoparticles with albumin site-specific modification on the surface of the lipid nanoparticles. For details, see Figure 1 .

[0054] The albumin site-modified lipid nanoparticles disclosed in the present invention are prepared by lipid nanoparticles subjected to albumin site-modification, and the albumin site-modified drug-loaded lipid nanoparticles are prepared by drug-loaded lipid nanoparticles subjected to albumin site-modification. The lipid nanoparticles are prepared by using a functionalized low molecular weight PEG-maleimide derivative as a lipid compound and existing conventional lipid raw materials, the drug-loaded drug includes nucleic acid, and the existing conventional lipid raw materials are ionizable cationic lipids, PEGylated lipids, cholesterol and phospholipid derivatives. The carrier of the present invention includes a functionalized low molecular weight PEG lipid compound, and commonly used nucleic acid delivery composition materials: ionizable cationic lipids, PEGylated lipids, cholesterol and phospholipid derivatives; the nucleic acid is selected from one or more of mRNA, circular RNA, siRNA, microRNA, and antisense nucleic acid.

[0055] In the present invention, the lipid compound is a functionalized low molecular weight PEG-maleimide derivative, and its structure is as shown in formula (1): (1) Wherein, R1 and R2 are -C(=O)OR, wherein R is an unsubstituted C8-C 24 Straight chain alkyl, unsubstituted C6-C 18 branched alkyl, straight chain alkyl substituted by C3-C8 cycloalkyl, or unsubstituted C8-C 24 Cycloalkyl; X is an amino acid residue; Y contains an ethylene glycol segment and also contains an amide group and / or an amino acid residue; Mal is a maleimide group; n is 1 to 3.

[0056] In the present invention, the functionalized low molecular weight PEG lipid compound accounts for 1% to 35% (mol / mol) of the existing conventional lipid raw materials; the functionalized low molecular weight PEG lipid compound is selected from one or more of maleimide lipid compounds and maleimide derivatives, such as maleimide lipid compounds. Because it can undergo Michael addition reaction with the only thiol group of albumin, it can perform site-specific modification on the surface of lipid nanoparticles. As an example, it includes Mal-PEG4-G-DMG, Mal-PEG8-G-DMG, Mal-PEG 12 -G-DMG, Mal-PEG 16 -G-DMG, Mal-PEG4-VC-PAB-G-DMG, (Mal-PEG4)2-Lys-DMG or one or more thereof.

[0057] In the present invention: ionizable cationic lipids include but are not limited to one or more of SM-102, ALC-0315, DLin-MC3-DMA, cKK-E12, L319, DLin-KC2-DMA, C12-200, DOTAP, 306Oi10, which account for 30% to 70% (mol / mol), preferably 40% to 60% (mol / mol) of the existing conventional lipid raw materials; Phospholipid derivatives include, but are not limited to, one or more of DSPC, DOPC, DPPC, POPC, DOPE, DSPE, and DPPE, which account for 2% to 15% (mol / mol), preferably 5% to 15% (mol / mol) of the existing conventional lipid raw materials; Cholesterol (including its derivatives) includes but is not limited to: one or more of β-sitosterol, cholestanol, cholestanone, cholesterol, cholestenone, 7β-hydroxycholesterol, 7α-hydroxycholesterol, which accounts for 20% to 45% (mol / mol) of the existing conventional lipid raw materials, preferably 30% to 40% (mol / mol); The PEGylated lipids include, but are not limited to, one or more of DMG-PEG, DSG-PEG, DPG-PEG, and DSPE-PEG, which account for 0.3% to 20% (mol / mol), preferably 0.8% to 10% (mol / mol) of the existing conventional lipid raw materials.

[0058] The molar amount of existing conventional lipid raw materials is the sum of the molar amounts of ionizable cationic lipids, phospholipid derivatives, cholesterol, and PEGylated lipids.

[0059] In the present invention, the mass ratio of the ionizable cationic lipid to the nucleic acid is (1-50):1; preferably (5-30):1; and more preferably (10-20):1.

[0060] In the present invention, the hydrated particle size of the lipid nanoparticles is 10 to 120 nm, preferably 80 to 100 nm.

[0061] In the present invention, the encapsulation rate of nucleic acid in the drug-loaded lipid nanoparticles is 30% to 99%, preferably 80% to 99%.

[0062] In the present invention, the method for preparing lipid nanoparticles modified with albumin at a specific site comprises the following steps: preparing lipid nanoparticles by physically mixing an aqueous phase with a lipid organic phase or by a microfluidic method.

[0063] In the present invention, the preparation method of albumin site-specifically modified drug-loaded lipid nanoparticles comprises the following steps: a nucleic acid-containing aqueous phase and a lipid organic phase are mixed by physical mixing or microfluidics to prepare nucleic acid-loaded lipid nanoparticles.

[0064] The aqueous phase includes water-soluble drugs and buffer solutions; the lipid organic phase includes lipid compounds, conventional ionizable cationic lipids, phospholipid derivatives, cholesterol, pegylated lipids, and alcohol solvents.

[0065] Specifically, a water-soluble drug, such as nucleic acid (mRNA), is dissolved in a buffer solution as the aqueous phase. As an example, a water-soluble drug, such as nucleic acid (mRNA), is dissolved in a citrate buffer solution to a final concentration of 10 to 300 μg / mL, preferably 30 to 250 μg / mL.

[0066] Specifically, the organic phase uses various organic solvents such as small molecule alcohol solvents (such as ethanol) to dissolve the carrier materials (functionalized low molecular weight PEG lipid compounds, and commonly used ionizable cationic lipids: phospholipid derivatives: cholesterol: PEGylated lipids, and the molar ratio of the latter four raw materials is 50:10:38.5:1.5); among them, the low molecular weight PEG lipid compounds account for 1.5%~6% of the latter four raw materials, calculated by molar amount.

[0067] In the present invention, cationic lipids can be ionized under acidic conditions and protonated to form positively charged lipids, which are combined with negatively charged nucleic acids by electrostatic interaction to form nucleic acid-carrying nanoparticles.

[0068] As an example, the physical mixing method includes the following steps: aspirating the aqueous phase containing nucleic acid and adding it into the lipid organic phase in a vortex state, mixing and then letting it stand, so as to obtain the nucleic acid-loaded lipid nanoparticles.

[0069] Optionally, the product obtained by the physical mixing method is dialyzed, for example, dialyzed in a PBS buffer solution (11.8 mM, pH 7.4) with a volume greater than 1000 times for more than 4 hours.

[0070] The microfluidic method comprises the following steps: using a syringe to draw a lipid organic phase (lipid concentration: 3-15 mg / mL) and an aqueous phase containing (50-200 μg / mL) nucleic acids respectively, and injecting the lipid organic phase and the aqueous phase into a microfluidic chip at a flow rate of 1:3 for mixing, and stopping collecting the liquid after the aqueous phase solution in the syringe is emptied to obtain lipid nanoparticles carrying nucleic acids; preferably, the microfluidic method also includes dialyzing the obtained mixed solution, such as dialyzing in PBS (11.8 mM, pH=7.4) with a volume greater than 1000 times its volume for more than 4 hours.

[0071] The lipid nanoparticles or drug-loaded lipid nanoparticles prepared by the present invention are mixed with an albumin solution and then incubated to obtain lipid nanoparticles or drug-loaded lipid nanoparticles with albumin coupled to the surface; specifically, the lipid nanoparticles or drug-loaded lipid nanoparticles are mixed with an albumin solution and then incubated at room temperature, and then placed at 4°C to obtain lipid nanoparticles or drug-loaded lipid nanoparticles with albumin coupled to the surface, preferably placed at 4°C for 10 to 50 hours, and more preferably placed at 4°C for 12 to 30 hours; as an example, the lipid nanoparticles or drug-loaded lipid nanoparticles are mixed with an albumin solution and then incubated at room temperature, placed at 4°C for 10 to 30 hours, and then PBS (11.8 mM, pH=7.4) is used as the mobile phase, and agarose gel column chromatography is performed to obtain lipid nanoparticles mRNA-LNPs with nucleic acid coupled to the surface of albumin. The steric hindrance brought by the surface coupling of albumin can reduce or overcome the immunogenicity of the existing nanoparticles covered with PEG on the outer layer to the body.

[0072] The present invention discloses the use of the mRNA-LNP containing a functionalized low molecular weight PEG lipid compound as a biological product. The biological product is a vaccine, preferably an mRNA vaccine. Including but not limited to: influenza vaccine, AIDS vaccine, viral pneumonia vaccine, tuberculosis vaccine, respiratory syncytial virus vaccine, enterovirus vaccine, intestinal mucosa-associated tumor vaccine or lung cancer vaccine, etc.

[0073] The biological product is used to prevent and / or treat infectious diseases or tumors, including but not limited to melanoma, colon cancer and other tumors. The biological product can be used by injection, oral administration, external application and other methods.

[0074] The present invention provides a lipid compound, which is a functionalized low molecular weight PEG-maleimide derivative, and its exemplary chemical structure is as follows (2): (2)

[0075] Wherein, X is a polypeptide amino acid chain, comprising different types of amino acid residues and PEG fragments.

[0076] In a preferred embodiment of the present invention, the lipid compound chemical structure contains PEG functionalized linkers of different chain lengths, namely: MC-PEG4-DMG, MC-PEG8-DMG, MC-PEG 12 -DMG, MC-PEG 16 -DMG, MC-PEG4-VC-PNP-G-DMG MC-PEG4-Lys(MC-PEG4)-DMG; their chemical structures are respectively as follows: (3) (4) (5) (6) (7) (8) Unless otherwise specified, MC-PEG 16 -DMG (chemical formula 6) lipid compound has outstanding biological effects in in vitro cell transfection and in vivo lymphatic distribution, transfection and other studies.

[0077] The above-mentioned functionalized low molecular weight PEG-maleimide derivative is combined with commonly used nucleic acid delivery lipid composition materials and nucleic acid drugs to prepare nucleic acid-loaded lipid nanoparticles. The maleimide on the surface of the nanoparticles is chemically coupled with the only thiol group of albumin through Michael addition reaction, thereby obtaining nucleic acid-loaded lipid nanoparticles mRNA-LNP with albumin site-specifically coupled on the surface.

[0078] The following specific implementation methods and examples are used to illustrate the present invention, but do not limit the scope of the present invention. The raw materials used are existing products, and the specific preparation operations, performance tests, and data analysis are all conventional techniques. For example, TLC monitoring reactions use G-type silica gel plates, petroleum ether: ethyl acetate = 10:1, and ultraviolet color development; the column chromatography filler is 200 mesh silica gel H type.

[0079] The animal experiments complied with the relevant requirements of Soochow University.

[0080] Nucleic acid information: EGFP mRNA (encoding enhanced green fluorescent protein), Luc mRNA (encoding luciferase), OVA mRNA (encoding chicken ovalbumin) are commonly used functional mRNAs and are commercially available. Albumin is human serum albumin; dichlororesin was purchased from Nankai Hecheng Technology Co., Ltd.

[0081] 1. Synthesis of functionalized low molecular weight PEG-maleimide derivatives (lipid compounds) See also Figure 2 , intermediate B: 4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane is synthesized using commercial product A as a raw material; intermediate C: 3-benzyloxy-1,2-propanediol is synthesized using intermediate B as a raw material; intermediate D: 3-benzyloxy-1,2-propanediol tetracosanoate is synthesized using intermediate C as a raw material; intermediate DMG: 1,2-dimyristyl glycerol is synthesized using intermediate D as a raw material; intermediate E: 3-(9-fluorenylmethoxycarbonylglycyloxy)propane-1,2-diyl tetracosanoate is synthesized using intermediate DMG and commercial product Fmoc-Gly-OH as raw materials; intermediate DMD-G: 1,2-tetradecanoylglycerol-3-glycine is synthesized using intermediate E as a raw material.

[0082] See also Figure 3 and Figure 4 Using Fmoc-PEG4-OH and 6-maleimidocaproic acid as raw materials, the intermediates Mal-PEG4-OH, Mal-PEG8-OH, and Mal-PEG 12 -OH, Mal-PEG 16 -OH, the intermediates synthesized above can be uniformly expressed as Mal-PEG n -OH.

[0083] See also Figure 5 , using the intermediate Mal-PEG n -OH and the intermediate DMG-G were used as raw materials to synthesize the lipid compound Mal-PEGn-DMG.

[0084] The lipid compound (functionalized low molecular weight PEG-maleimide derivative, Mal-PEGn-DMG) and the commonly used nucleic acid delivery lipid composition material (ionizable cationic lipid: phospholipid derivative: cholesterol: PEGylated lipid, the molar ratio of which is 50:10:38.5:1.5) are dissolved in an organic solvent such as ethanol as an organic phase; the nucleic acid (mRNA) is dissolved in a citrate buffer solution as an aqueous phase; the above lipid organic phase and the aqueous phase containing nucleic acid are prepared by physical mixing or microfluidics to obtain the nucleic acid-carrying lipid nanoparticle mRNA-LNP. Then, it is mixed with an albumin solution, incubated at room temperature in turn, and placed at 4°C overnight to complete the coupling of the thiol group on the albumin with the maleimide on the surface of the nanoparticle, and then separated and purified by agarose gel column chromatography to obtain: nucleic acid-carrying lipid nanoparticles HSA-mRNA-LNP with fixed-point coupling of albumin on the surface.

[0085] Embodiment 1 1. Preparation of intermediate B: 4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane Weigh 515 mg of NaH in a double-necked flask, add 10 mL of DMF under nitrogen atmosphere and atmospheric pressure, add commercially available glycerol acetone (compound A, 1.0 g) in an ice-water bath, stir conventionally, add α-bromomethylbenzene, stir at room temperature, and after TLC monitoring, add 10 mL of ice water to the reaction system to quench the reaction, extract with ether (60 mL×3), combine the organic phases, and wash with saturated NaCl water 3 times, concentrate the organic phase under reduced pressure, and separate by column chromatography to obtain 1.3 g of compound B (yield 77.33%). Hydrogen spectrum identification: 1 H NMR (400 MHz, CDCl3) δ 7.34 (s, 5H), 4.58 (d, J = 6.0 Hz,2H), 4.31 (p, J = 6.1 Hz, 1H), 4.06 (dd, J = 8.3, 6.4 Hz, 1H), 3.75 (dd, J =8.3, 6.3 Hz, 1H), 3.55 (d, J = 5.7 Hz, 1H), 3.50 – 3.45 (m, 1H), 1.42 (s,3H), 1.37 (s, 3H). The above hydrogen spectrum data confirmed that the obtained compound was correct.

[0086] 2. Preparation of Intermediate C: 3-Benzyloxy-1,2-propanediol Weigh intermediate B (800 mg) into a single-mouth bottle, add 14 mL of acetic acid and 6 mL of water to dissolve, stir at 65°C for 1 hour, and cool naturally to room temperature; add saturated NaHCO3 aqueous solution to the reaction system, adjust to pH 7, extract with dichloromethane (60 mL×3), combine the organic phases, dry and concentrate, and purify by column chromatography to obtain 500 mg of compound C (yield 76.33%). Hydrogen spectrum identification: 1 H NMR (400 MHz, CDCl3) δ 7.39 – 7.28 (m, 5H), 4.55 (s, 2H), 3.89(ddd, J = 9.8, 5.8, 4.0 Hz, 1H), 3.76 – 3.67 (m, 2H), 3.66 – 3.58 (m, 1H),3.58 – 3.51 (m, 2H), 1.23 (t, J = 7.0 Hz, 1H). The above hydrogen spectrum data confirmed that the obtained compound was correct.

[0087] 3. Preparation of intermediate D: 3-benzyloxy-1,2-propanediol tetracosanoate Weigh intermediate C (500 mg), N,N'-dicyclohexylcarbodiimide (1700 mg) and 4-dimethylaminopyridine (56.5 mg) in a 50 mL single-mouth bottle, add 20 mL of dichloromethane to dissolve, stir at room temperature, add tetradecanoic acid (1880 mg) and stir at room temperature overnight. After TLC monitoring, the solid impurities in the reaction solution are removed by filtration, and one volume of water is added to the filtrate, and washed with dichloromethane 3 times. The organic phases are combined, dried and concentrated, and purified by column chromatography to obtain 1300 mg of compound D (yield 78.64%). Hydrogen spectrum identification: 1 H NMR (400 MHz, CDCl3) δ 7.32 (td, J = 6.8, 2.5Hz, 5H), 5.29 – 5.20 (m, 1H), 4.54 (d, J = 5.7 Hz, 2H), 4.34 (dd, J = 11.9,3.8 Hz, 1H), 4.19 (dd, J = 11.9, 6.4 Hz, 1H), 3.59 (dd, J = 5.2, 1.0 Hz, 2H), 2.30 (dt, J = 17.0, 7.6 Hz, 4H), 1.61 – 1.57 (m, 4H), 1.34 – 1.22 (m, 40H),0.90 – 0.85 (m, 6H). The above hydrogen spectrum data confirmed that the obtained compound was correct.

[0088] 4. Preparation of intermediate DMG: 1,2-dimyristyl glyceride Weigh the intermediate D (500 mg) into a 50 mL single-mouth bottle, add acetic acid / ethanol (6 mL:3 mL, 2:1) to dissolve, add Pd / C (400 mg, content 10%), replace with hydrogen three times, and stir at room temperature for 1 hour under a hydrogen atmosphere, dilute the reaction solution with dichloromethane, filter out the palladium carbon catalyst, dissolve the soluble matter in the solid with dichloromethane, combine the organic phase dichloromethane solution, wash once with a saturated NaHCO3 aqueous solution, wash three times with saturated NaCl water, dry and concentrate, and chromatograph on a silica gel column to obtain 350 mg of compound DMG (yield 82.35%). Hydrogen spectrum identification: 1 H NMR (400 MHz, CDCl3) δ 5.08 (dd, J = 10.2,4.9 Hz, 1H), 4.28 (ddd, J = 17.6, 11.9, 5.1 Hz, 2H), 3.73 (t, J = 5.4 Hz,2H), 2.33 (dt, J = 8.9, 7.6 Hz, 4H), 2.07 (s, 1H), 1.64 (dd, J = 14.4, 7.5Hz, 3H), 1.27 (d, J = 10.9 Hz, 41H), 0.88 (t, J = 6.9 Hz, 6H). The above hydrogen spectrum data confirmed that the obtained compound was correct.

[0089] 5. Preparation of intermediate E: 3-(9-fluorenylmethoxycarbonylglycyloxy)propane-1,2-diyltetracosanate Weigh the intermediate DMG (200 mg), commercially available Fmoc-Gly-OH (176 mg) and 4-dimethylaminopyridine (48 mg) in a 25 mL single-mouth bottle, add 10 mL of dichloromethane to dissolve, place the reaction system at 0°C, add N,N-dicyclohexylcarbodiimide (120 mg); after TLC monitoring of the reaction is complete, filter out solid impurities, add one volume of water to the filtrate, and wash with dichloromethane three times, combine the organic phases, dry and concentrate, and purify by column chromatography to obtain 253 mg of compound E (yield 82.14%). Hydrogen spectrum identification: 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d,J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (td, J = 7.4, 0.9 Hz, 2H), 5.29 (dd, J = 9.2, 4.8 Hz, 2H), 4.40 (dd, J = 11.0, 5.5 Hz, 3H), 4.33 – 4.21(m, 3H), 4.15 (dd, J = 11.9, 5.8 Hz, 1H), 4.01 (d, J = 4.9 Hz, 2H), 2.31 (t,J = 7.5 Hz, 4H), 1.63 – 1.57 (m, 4H), 1.26 (d, J = 9.9 Hz, 40H), 0.88 (t, J =6.8 Hz, 6H). The above hydrogen spectrum data confirmed that the obtained compound was correct.

[0090] 6. Preparation of intermediate DMG-G: 1,2-tetradecanoylglycerol-3-glycine Weigh compound E (50 mg) into a 25 mL single-mouth bottle, add 20 mL of dichloromethane to dissolve, place the reaction system at 0°C, add 11 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), stir for 0.5 hours, then add an equal volume of water to the reaction solution to dilute, extract with ethyl acetate (10 mL×3), dry and concentrate, and perform silica gel column chromatography to obtain 21 mg of compound DMG-G (yield 58.39%). Hydrogen spectrum identification: 1 H NMR (400 MHz, CDCl3) δ 5.31 – 5.24 (m, 1H), 5.08 (s, 1H), 4.28 (dd, J = 22.1, 5.1 Hz, 4H), 3.73 (d, J = 5.2 Hz,1H), 2.32 (dt, J = 7.7, 5.6 Hz, 4H), 1.64 (s, 4H), 1.27 (d, J = 11.2 Hz,40H), 0.88 (t, J = 6.8 Hz, 6H). The above hydrogen spectrum data confirmed that the obtained compound was correct.

[0091] Embodiment 2 1. Using Fmoc-PEG4-OH and 6-maleimidocaproic acid as raw materials, the intermediates Mal-PEG4-OH, Mal-PEG8-OH, and Mal-PEG12 -OH, Mal-PEG 16 -OH. MC-PEG 16 - Preparation of DMG is used as an example.

[0092] Dichlororesin (1.0 g) was weighed and added to the solid phase reaction column. After washing twice with dichloromethane, the resin was swollen with dichloromethane for 30 min and then the solvent was removed.

[0093] The first Fmoc-PEG4-OH (1.6 g) was dissolved in dichloromethane, and 0.6 mL N,N-diisopropylethylamine was added for activation. The solution was added to the solid phase reaction tube of the swollen resin, and stirred for reaction for 3 hours under nitrogen protection. After adding 30 mL MeOH to seal for 1 hour, it was washed with DMF for 4 times, shrunk by MeOH, and vacuum dried. A piperidine / DMF mixed solution (1:3, v / v) was added to remove Fmoc. The reaction was carried out twice, with reaction times of 5 min and 10 min respectively. After the deprotection was completed, it was washed with DMF and dichloromethane for 5 times each to obtain NH2-PEG4-dichloro resin.

[0094] The second amino acid analog Fmoc-PEG4-OH (1.6 g) and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL DMF, and N,N-diisopropylcarbodiimide (171 μL) was added for activation for 30 min under ice bath conditions. The solution was added to the solid phase reaction tube containing the resin in the previous step, and dimethylaminopyridine (27 mg) was added. The reaction was stirred for 3 hours under nitrogen protection, and then a piperidine / DMF mixed solution (1:3, v / v) was added to remove Fmoc. The reaction was repeated twice with reaction times of 5 min and 10 min respectively. After the deprotection was completed, the mixture was washed with DMF and dichloromethane 5 times each to obtain NH2-PEG4-PEG4-dichloro resin.

[0095] The third amino acid analog Fmoc-PEG4-OH (1.6 g) and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL DMF, and N,N'-diisopropylcarbodiimide (171 μL) was added for activation for 30 min under ice bath conditions. The solution was added to the solid phase reaction tube containing the resin in the previous step, and dimethylaminopyridine (27 mg) was added. The reaction was stirred for 3 hours under nitrogen protection, and then a piperidine / DMF mixed solution (1:3, v / v) was added to remove Fmoc. The reaction was carried out twice, with reaction times of 5 min and 10 min respectively. After the deprotection was completed, it was washed with DMF and dichloromethane 5 times each to obtain NH2-PEG4-PEG4-PEG4-dichloro resin.

[0096] The fourth amino acid analog Fmoc-PEG4-OH (1.6 g) and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL DMF, and N,N-diisopropylcarbodiimide (171 μL) was added for activation for 30 min under ice bath conditions. The solution was added to the solid phase reaction tube containing the resin in the previous step, and dimethylaminopyridine (27 mg) was added. The reaction was stirred for 3 hours under nitrogen protection, and then a piperidine / DMF mixed solution (1:3, v / v) was added to remove Fmoc. The reaction was carried out twice with reaction times of 5 min and 10 min, respectively. After the deprotection was completed, it was washed with DMF and dichloromethane 5 times each to obtain NH2-PEG4-PEG4-PEG4-PEG4-dichloro resin.

[0097] 6-Maleimidocaproic acid (234 mg) and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL DMF, and N,N'-diisopropylcarbodiimide (171 μL) was added for activation for 30 min under ice bath conditions. The solution was added to the solid phase reaction column containing the resin in the previous step, and dimethylaminopyridine (27 mg) was added. The reaction was stirred for 3 hours under nitrogen protection to obtain Mal-PEG. 16 -OH.

[0098] Mal-PEG 16 -OH was removed from the resin: the above resin was vacuum dried and transferred to a round-bottom flask, and removed with a trifluoroethanol (TFE)-dichloromethane mixed solution (1:4, v / v) for 2 hours, then filtered, and when the filtrate was concentrated to one-fourth of the original volume, the concentrate was added to 10 times the amount of ether for precipitation, and allowed to stand at 4°C for 2 hr., and then filtered again. The filter cake was washed with ether 6 times and vacuum dried to obtain a crude polypeptide powder.

[0099] Separation and purification: The crude peptide was separated and purified by high performance liquid chromatography. The conditions were: phase A was 0.1% trifluoroacetic acid aqueous phase, phase B was acetonitrile phase, phase A-phase B = 10%-90%, 20 min, and the peptide sample (purity ≥ 95%) was obtained, which was 253 mgMal-PEG 16 -OH. Mass spectrometry identification: MS-ESI (m / z): [MH]- calcd for: 1128.65; found: 1127.79. The above mass spectrometry data confirmed that the obtained compound was correct.

[0100] Referring to the above method, one Fmoc-PEG4-OH, two Fmoc-PEG4-OH, and three Fmoc-PEG4-OH were used to obtain Mal-PEG4-OH, Mal-PEG8-OH, and Mal-PEG 12 -OH.

[0101] 2. Use of intermediate Mal-PEG 16 -OH and intermediate DMG-G were used as raw materials to synthesize Mal-PEG 16 -DMG.

[0102] Weigh the compounds DMG-G (40 mg), Mal-PEG 16 -OH (43 mg) and 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (40 mg) were placed in a 25 mL single-mouth bottle, 10 mL of DMF was added to dissolve, and then the reaction system was placed in a 0°C environment, N,N-diisopropylethylamine (25 μL) was added, and after TLC monitoring of the reaction was complete, one volume of water was added to the reaction system, and the reaction system was washed with dichloromethane three times, the organic phases were combined, dried and concentrated, and 55 mg of Mal-PEG was obtained by silica gel column chromatography. 16 -DMG (yield 44%). Mass spectrometry identification: MS-ESI (m / z): [M+2H] 2+ calcd for : 1751.11 ; found:876.56. The above mass spectrum data confirmed that the obtained compound was correct.

[0103] Referring to the above method, the intermediate Mal-PEG n -OH and intermediate DMG-G were used as raw materials to synthesize Mal-PEGn-DMG. Mass spectrometry data confirmed that the obtained compound was correct.

[0104] Embodiment 3 1. Preparation and characterization of nucleic acid-loaded lipid nanoparticles Ionizable cationic lipids can be protonated under acidic conditions to form positively charged lipids, which can bind to negatively charged nucleic acids through electrostatic interaction to form nucleic acid-carrying lipid nanoparticles mRNA-LNP. In the mRNA-LNP prepared by the present invention, the mass ratio of ionizable cationic lipids to encapsulated nucleic acids is (10-30):1, the molar ratio of ionizable cationic lipids: auxiliary phospholipids: cholesterol: polyethylene glycol lipids is 50:10:38.5:1.5, and the ionizable cationic lipid is SM-102.

[0105] Note: The chemical name of SM-102 is: 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester; cholesterol; auxiliary phospholipid is DSPC; polyethylene glycol lipid DMG-PEG2000; the prepared product is a 4-component nucleic acid-loaded lipid nanoparticle mRNA-LNP.

[0106] The five-component lipid nanoparticle HSA-mRNA-LNP of the present invention is prepared by adding Mal-PEG4-DMG (accounting for 1.5%, 3.0%, 6.0%, and 12% respectively) to the above four components. The addition ratio of Mal-PEGn-DMG in HSA-mRNA-LNP is based on the relative molar ratio of the four components of lipids.

[0107] Preparation method: mRNA was dissolved in 50 mM sodium citrate buffer solution (pH 5.0) to a final concentration of 200 μg / mL (aqueous phase). SM-102: cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5 molar ratio was mixed to form a lipid mixture (oil phase). A certain molar ratio of Mal-PEG was added to the four components. 16 -DMG lipids (1.5%, 3.0%, 6.0%, 12%) were used to form five-component liposomes, each of which was dissolved in ethanol to form an organic phase; the flow rates of the aqueous phase and the oil phase were controlled by microfluidics to mix the mRNA and lipid mixture in a volume ratio of 3:1, and the buffer environment was replaced with PBS at pH 7.4 by dialysis or tangential flow to remove ethanol, and four types of mRNA-LNPs were prepared, namely: PEG 16 -LNP (1.5%) PEG 16 -LNP (3.0%) PEG 16 -LNP (6.0%) PEG 16 -LNP (12%) .

[0108] The product of only four components is Moderna-LNP.

[0109] Referring to the above method, nucleic acid-loaded nanoparticles PEGn-LNP were prepared using Mal-PEGn-DMG.

[0110] 2. Preparation and characterization of albumin-modified nucleic acid-loaded lipid nanoparticles 10 mg / mL albumin solution (40 μL, 80 μL, 160 μL, 320 μL) was added to PEGn-LNP prepared with Mal-PEGn-DMG with different PEG units, incubated at room temperature for 2 hours, placed at 4°C for 20 hours, and separated and purified with agarose gel filtration column using PBS (11.8 mM, pH=7.4) as the mobile phase to obtain a solution of five-component nucleic acid-loaded lipid nanoparticles HSA-mRNA-LNP coupled with albumin. After concentration using a 100K Millipore ultrafiltration centrifuge tube, the following were obtained: HSA-mRNA-LNP nucleic acid-loaded lipid nanoparticles modified with different amounts of albumin containing different PEG units. Since each nanoparticle needs to reflect more information, it needs to be abbreviated. For example: Mal-PEG 16 -DMG accounts for 1.5% of the nanoparticles, which can be abbreviated as HSA-PEG 16 -LNP （1.5%） .

[0111] The final product of nanoparticles prepared with different prescriptions was diluted 10 times with diluent, added into the particle size pool 1 mL, and placed on the Malvern ZetaSizer instrument to detect the characterization data of hydrated particle size, polydispersity index, and surface potential, as shown in Table 1. Among them, the mRNA is EGFP mRNA, the mass ratio of ionizable lipids to mRNA is 20:1, and the albumin solution is 160 μL.

[0112] The nucleic acid encapsulation efficiency of each nanoparticle was determined using a Ribogreen kit and 1% Triton as a demulsifier, and the results were 90-94%.

[0113] Table 1 Hydrated particle size, polydispersity index and surface potential of nanoparticles

[0114] The surface potential column in Table 1: the four-component Moderna-LNP is -1.0 mV; the five-component PEG 16 -LNP is -1.4mV; 5-component HSA-PEG 16 -LNP is -5.7mV; the potential of free human serum albumin HSA is -13mV. It can be seen that 1) HSA has a large negative charge, and the five-component PEG 16 -LNP has a small negative charge, while HSA-PEG 16 -LNP negative charge increases, and the fifth component Mal-PEG in the nanoparticles 16 -DMG is added and coupled to HSA; 2) HSA-PEG 16The albumin coupled to the LNP surface at a fixed point can form a spatial shielding effect on the PEG on its surface, thereby promoting the reduction of the immunogenicity of PEG on the surface of the nanoparticles, which is one of the obvious technical advances achieved by the present invention.

[0115] Embodiment 4 1. In vitro cell transfection ability of nanoparticles Referring to the above method, EGFP mRNA (mRNA expressing green fluorescent protein) was used as the encapsulated nucleic acid to prepare HSA-PEGn-LNP loaded with EGFP mRNA, with a mass ratio of 20:1 and an albumin solution of 160 μL. The experimental groups were set as follows: HSA-PEG n -LNP (1.5%) , HSA-PEG n -LNP (3.0%) , HSA-PEG n -LNP (6.0%), HSA-PEG n -LNP (12%) The control group was Moderna-LNP (four-component nanoparticles, benchmarked against commercially available products), and the same experimental operations were performed to evaluate the cell transfection ability of lipid nanoparticles. The day before the experiment, DC2.4 cells were plated, and 50,000 cells were seeded in each well of a 48-well cell culture plate. When the cell confluence was 70%-80%, the above groups of nanoparticles were added, and the mixture was gently shaken and cultured in a saturated humidity incubator at 37°C and 5% CO2 for 3 hours. The cells were digested and separated with trypsin to obtain a single cell suspension, and the average fluorescence intensity of the cells was detected by flow cytometry to verify the in vitro cell transfection efficiency of each group of nanoparticles. The results are shown in Table 2.

[0116] Table 2 Cell transfection ability of nanoparticles

[0117] The transfection status of DC2.4 cells can be seen: HSA-PEG in the experimental group 16 -LNP (fluorescence value is 2746), control group PEG 16 -LNP (fluorescence value is 1269), the experimental group increased by 2.2 times. 16 -LNP has the strongest ability to transfect dendritic cells.

[0118] 2. Investigation of the lymph node targeting ability of nanoparticles in animals PEG n -LNP (3.0%) , HSA-PEG n -LNP (3.0%), as the experimental group, and the commercially available Moderna-LNP (four-component nanoparticles, benchmarked against commercially available products) as the control group to investigate lymph node targeting.

[0119] The fluorescently labeled LNP nanoparticles were prepared by adding 300 μg of near-infrared fluorescent dye DiR to the lipid organic phase, which is a conventional technique. LNP (DiR dosage was 50 ng / kg) was injected subcutaneously at the tail base of 6-8 week-old female C57BL / 6 mice. The mice were killed 6 hours later, and the left and right inguinal lymph nodes were removed. After washing with saline, the water was dried with filter paper, and the fluorescence intensity was detected using a small animal in vivo imaging system. The results are detailed in Figure 6 , Table 3.

[0120] Table 3 Specific fluorescence values ​​of nanoparticles enriched in lymph nodes

[0121] As can be seen from Table 3, lymph node targeting ability: HSA-PEG 16 -LNP is significantly better than PEG n -LNP and commercially available Moderna-LNP, and HSA-PEG 16 -LNP （3.0%） The lymph node targeting is the strongest.

[0122] 3. Tumor inhibition experiment of nanoparticles on melanoma According to the method of Example 2, LNP loaded with chicken ovalbumin OVA-mRNA was prepared, and each group was arranged as follows: PBS solution was used as a negative control group, free OVA-mRNA was used as a positive control group 1, Moderna-LNP (four-component nanoparticles, benchmarked against commercially available products) was used as a positive control group 2, and PEG 16 -LNP (3.0%) As the positive control group 3, HSA-PEG 16 -LNP (3.0%) The experimental group (mass ratio was 20∶1, albumin solution was 160 μL). The mice were subcutaneously inoculated with B16-OVA cell line. On the third day after tumor implantation, the prepared nanoparticles or PBS solution were injected into the hind leg muscle of tumor-bearing C57BL / 6 mice, with a dose of 20 μg mRNA. The second booster injection was performed on the 14th day. The tumor formation events of mice were recorded daily, and the length and width of the tumor were measured with a vernier caliper every other day. The tumor volume calculation formula is: tumor volume (cm 3 ) = tumor long diameter × tumor short diameter 2×0.5. Note: OVA-mRNA is the messenger RNA (mRNA) encoding ovalbumin (OVA). Ovalbumin is a large and complex glycoprotein that can induce a moderate immune response in the body after being degraded into specific antigenic peptides in cells. Therefore, it is widely used as a model antigen in immunology and biochemistry research.

[0123] The results showed that after treatment, HSA-PEG 16 -LNP （3%） Compared with the control group, the tumor volume was significantly reduced. Figure 7 , Table 4.

[0124] Table 4 Tumor volume of mice after 15 days of nanoparticle treatment (n = 7)

[0125] It can be seen that HSA-PEG 16 -LNP has obvious anti-tumor effect, HSA-PEG 16 -LNP （3%） The anti-tumor ability is significantly better than that of commercially available Moderna-LNP and PEG 16 -LNP （3%） .

[0126] 4. Investigation of Nanoparticle Activation of Dendritic Cells (1) Extraction and culture of dendritic cells (DCs) Under sterile conditions, tibia and femur of C57BL / 6 mice were taken and other tissues on the bones were removed. The two ends of the bones were cut off, and RPMI 1640 medium (without cytokines) was drawn with a syringe to repeatedly rinse the bones until the bone shaft turned white. The rinsing fluid was collected in a centrifuge tube and centrifuged at 1500rpm for 5min. The precipitate was resuspended in RPMI 1640 medium containing cytokines GM-CSF (20 ng / mL), IL-4 (10ng / mL), fetal bovine serum (10%, v:v), penicillin (100 U / mL), and streptomycin (100 μg / mL) and added to the cell culture flask, and cultured in an incubator at 37℃ and 5% CO2. The day of cell extraction was recorded as day 0, and half of the medium was replaced on the 3rd and 5th days, respectively. The suspended cells were collected on the 7th day, which were primary DCs (bone marrow-derived dendritic cells BMDC).

[0127] (2) Evaluation of the rate of maturation of dendritic cells DCs were treated with nanoparticles to examine the expression of CD80 and CD86. Specifically, DCs cultured for 7 days were taken and 1×10 6The cells were seeded in a 6-well plate and added with: PBS (negative control group); OVA protein-expressing mRNA (OVA-mRNA) as a single mRNA control (similar to the free group, positive control group 1); commercially available Moderna-LNP (positive control group 2); PEG 16 -LNP (3.0 %) (Positive control group 3); HSA-PEG 16 -LNP (3.0%) (Experimental group), the mRNA concentration was 2 μg / well.

[0128] After incubation at 37°C for 24 hours, the cells were collected by centrifugation and washed twice with PBS. The expression of DCs surface activation markers CD80 and CD86 was detected by flow cytometry. The data are shown in Table 5. It can be seen that the expression of DCs surface activation markers CD80 and CD86: PBS group did not change significantly, while HSA-PEG 16 -LNP (3.0%) After treatment of DCs, the expression of CD80 and CD86 increased significantly (see Table 5 for details). The maturation rate was 2.7 times that of Moderna-LNP and PEG 16 -LNP (3.0 %) The effect of OVA-mRNA on the expression of PBS was 2.22 times that of PBS.

[0129] The results showed that HSA-PEG 16 -LNP (3.0%) It can significantly stimulate the maturation of DCs. The higher the maturation rate of activated dendritic cells, the stronger the effect of activating T cells, and the more beneficial it is for immunotherapy.

[0130] Table 5 Results of nanoparticle activation of dendritic cells

[0131] The present invention dissolves a lipid compound (functionalized low molecular weight PEG-maleimide derivative, Mal-PEGn-DMG) and a commonly used nucleic acid delivery lipid composition material (ionizable cationic lipid, phospholipid derivative, cholesterol, pegylated lipid, etc.) in an organic solvent as an organic phase; dissolves a nucleic acid (such as mRNA) in a buffer solution as an aqueous phase; and prepares the nucleic acid-carrying lipid nanoparticle mRNA-LNP by a physical mixing method or a microfluidic method by mixing the above lipid organic phase with the aqueous phase containing nucleic acid. Then, the mixture is mixed with an albumin solution, incubated at room temperature in turn, and placed at 4°C overnight to complete the coupling of the thiol group on the albumin with the maleimide on the surface of the nanoparticle, and then separated and purified by agarose gel column chromatography to obtain the nucleic acid-carrying lipid nanoparticle HSA-mRNA-LNP with fixed-point coupling of albumin on the surface. The steric hindrance brought by the surface coupling of albumin can reduce or overcome the immunogenicity of the existing nanoparticle outer layer covered with PEG to the body.

Claims

1. An albumin-site-modified lipid nanoparticle or an albumin-site-modified drug-loaded lipid nanoparticle, characterized in that: Includes lipid nanoparticles and albumin on their surface.

2. The albumin site-specifically modified drug-loaded lipid nanoparticles according to claim 1, characterized in that: The drug includes nucleic acid.

3. The albumin site-specific modified lipid nanoparticles or albumin site-specific modified drug-loaded lipid nanoparticles according to claim 1, characterized in that: The raw materials for preparing lipid nanoparticles include lipid compounds; the lipid compounds contain hydrophobic segments, hydrophilic segments and groups that can be coupled with proteins; the hydrophobic segments and the groups that can be coupled with proteins are respectively located at the ends of the lipid compounds.

4. The albumin site-specific modified lipid nanoparticles or albumin site-specific modified drug-loaded lipid nanoparticles according to claim 3, characterized in that: In the lipid compound, the hydrophobic segment includes an alkyl segment; the hydrophilic segment includes an ethylene glycol segment, a hydrophilic functional polypeptide segment or a PEG-like segment; and the group that can be coupled to a protein includes a maleimide group.

5. The albumin site-specific modified lipid nanoparticles or the albumin site-specific modified drug-loaded lipid nanoparticles according to claim 4, characterized in that: The chemical structural formula of the lipid compound is as follows: ; Among them, R1 and R2 are -C(=O)OR respectively, wherein R is a substituted or unsubstituted alkyl group; X is the residue of a compound containing an amino group and a carboxyl group after substitution; Y includes an ethylene glycol segment, a hydrophilic functional polypeptide segment or a PEG-like segment; Mal is a maleimide group; and n is 1 to 5.

6. A lipid compound, characterized in that The lipid compound contains a hydrophobic segment, a hydrophilic segment and a group that can be coupled with a protein; the hydrophobic segment and the group that can be coupled with a protein are respectively located at the ends of the lipid compound.

7. The method for preparing the albumin site-specific modified lipid nanoparticles or the albumin site-specific modified drug-loaded lipid nanoparticles according to claim 1, characterized in that: Lipid nanoparticles are prepared using lipid nanoparticle raw materials as raw materials, and then albumin is modified on the lipid nanoparticles to obtain lipid nanoparticles with site-specific modification of albumin; drug-loaded lipid nanoparticles are prepared using lipid nanoparticle raw materials and drugs as raw materials, and then albumin is modified on the drug-loaded lipid nanoparticles to obtain drug-loaded lipid nanoparticles with site-specific modification of albumin.

8. The method for preparing the albumin site-specific modified lipid nanoparticles or the albumin site-specific modified drug-loaded lipid nanoparticles according to claim 7, characterized in that: The lipid nanoparticle raw materials include the lipid compound described in claim 6 and other raw materials; the molar ratio of the lipid compound to other raw materials is 1% to 35%; the lipid nanoparticles are mixed and incubated with albumin to obtain lipid nanoparticles with albumin site-specific modification; the drug-loaded lipid nanoparticles are mixed and incubated with albumin to obtain drug-loaded lipid nanoparticles with albumin site-specific modification.

9. Use of the albumin site-specifically modified lipid nanoparticles or the albumin site-specifically modified drug-loaded lipid nanoparticles according to claim 1, or the lipid compound according to claim 6 in the preparation of biological products or drugs.

10. A biological product or drug, the active ingredient of which comprises the albumin site-specifically modified drug-loaded lipid nanoparticles according to claim 1.

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