Albumin site-directed modified lipid nanoparticles and preparation and application thereof

Abstract

The application discloses albumin site-directed modification lipid nanoparticles and preparation and application thereof, and relates to a kind of albumin site-directed modification lipid nanoparticles, which is prepared by synthesizing functionalized low molecular weight polyethylene glycol (PEG) lipid compound-maleimide derivative, together with the nucleic acid delivery lipid composition material usually used, to prepare nucleic acid-loaded lipid nanoparticles, then make the maleimide on its surface couple with the thiol group of albumin, to prepare the lipid nanoparticles with albumin site-directed modification on the surface.The application reduces the immunogenicity generated by the existing mRNA-loaded lipid nanoparticles outer layer covered PEG to the body, improves the related immune organ targeting of the nanoparticles, effectively delivers mRNA to lymph nodes to induce anti-tumor effect, and lays a foundation for further developing it as a new type of vaccine that can activate the immune system to produce anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a nanocarrier and nanodrug system, specifically to an albumin-modified lipid nanoparticle, a drug-loaded lipid nanoparticle, its preparation method, and its application in mRNA delivery and anti-tumor activity. Background Technology

[0002] Messenger ribonucleic acid (mRNA) is transcribed from a DNA template, carrying genetic information encoding a target protein. It can be translated into a functional protein by ribosomes. This can be utilized to deliver exogenous mRNA into cells to obtain the desired 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 groundbreaking progress. Currently, tumor vaccines and other immunotherapies are considered very promising methods for treating malignant tumors. Tumor vaccines can be designed to target antigens specifically expressed by tumor cells, such as growth factors, or to target neoantigens generated by mutations in tumor cells. Cancer mRNA vaccines are primarily used for treatment, aiming to stimulate the body's immune response, particularly T-cell-mediated immune responses, to eliminate or reduce the number of tumor cells.

[0004] Because mRNA is unstable under physiological conditions, possesses immunogenicity, and carries the same negative charge as the cell membrane (making it difficult to enter cells and exert its effects), research on mRNA vaccines primarily focuses on two areas: delivery systems and the selection of tumor antigens. Viral vectors, due to their efficient transfection and expression capabilities, were once important tools for achieving efficient expression of exogenous genes. However, due to potential immune responses, lack of specificity, and limitations in mRNA length, they have gradually been replaced by non-viral vectors, such as cationic lipid nanoparticles, which have become the mainstream choice for gene delivery research.

[0005] Currently, the two commercially available mRNA vaccines (from Pfizer / BioNTech and Moderna) both use lipid nanoparticles (LNPs) as their delivery system. Both have a particle size of 80-100 nm and very similar compositions, containing the following four components: 1) ionizable cationic lipids; 2) PEGylated lipids; 3) cholesterol; and 4) phospholipid derivatives (e.g., distearate phosphatidylcholine, DSPC). Significant progress has been made in modifying lipid components and optimizing for specific cells or tissues. For example, CN116350757A demonstrates enhanced CAR-T cell generation in vivo by binding to CD5 antibodies; while CN117015374A prepares novel LNPs by adjusting lipid components to improve delivery efficiency. In addition, CN115925975A constructed a (PA)2 peptide nanomicelle carrying the CD133-CAR plasmid and modified it with citric anhydride-modified dextran, a targeting group of the macrophage-specific target CD206. This vector enables CAR editing of macrophages in vitro and in vivo. Nevertheless, the performance of LNPs delivering mRNA still has significant room for improvement in terms of biocompatibility and transfection efficiency, including: reducing the immunogenicity of existing vaccines (mRNA-loaded lipid nanoparticles) coated with PEG, and improving vaccine targeting to deliver mRNA to relevant immune organs (lymph nodes), thereby activating the immune system to produce anti-tumor effects. Summary of the Invention

[0006] To address the shortcomings of existing nucleic acid-loaded (e.g., mRNA) lipid nanoparticles in terms of insufficient targeting of lymph nodes and the immunogenicity issues caused by the PEG chains on their surface, this invention provides a functionalized low molecular weight PEG-maleimide lipid compound. This compound is combined with commonly used nucleic acid delivery lipid composition materials (other conventional lipid nanoparticle raw materials) to prepare nucleic acid-loaded lipid nanoparticles. Through a Michael addition reaction, the maleimide on the surface of the nanoparticles is chemically coupled with the unique thiol group of albumin, thereby obtaining nanoparticles with albumin site-specific modification on the surface of the lipid nanoparticles.

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

[0008] An albumin-modified lipid nanoparticle, comprising the lipid nanoparticle and albumin on its surface.

[0009] An albumin-modified drug-loaded lipid nanoparticle comprises the aforementioned albumin-modified lipid nanoparticle and a drug.

[0010] In this invention, the albumin-modified lipid nanoparticles are prepared from lipid compounds and existing lipid composition materials.

[0011] This invention discloses a lipid compound containing a hydrophobic segment, a hydrophilic segment, and a protein-coupled group; the hydrophobic segment and the protein-coupled group are 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 couple with the protein includes a maleimide group. Specifically, the maleimide is at the hydrophilic tail, and the alkyl chain is intact, serving as the hydrophobic tail.

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

[0014] Specifically, the chemical structural formula of the lipid compound is as follows:

[0015] ;

[0016] Wherein, R1 and R2 are -C(=O)OR, where R is a substituted or unsubstituted alkyl group; X is the residue portion 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.

[0017] In this invention, the substituents in the substituted alkyl groups include alkyl groups, halogen groups, nitro groups, etc.

[0018] In this invention, alkyl groups include straight-chain alkyl groups, branched-chain alkyl groups, and cycloalkyl groups.

[0019] In this invention, the alkyl group has 1 to 30 carbon atoms; preferably, the alkyl group has 3 to 27 carbon atoms; more preferably, the alkyl group has 6 to 25 carbon atoms; as an example, the alkyl group includes unsubstituted C8-C atoms. 24 Straight-chain alkyl, unsubstituted C6-C 18 Branched alkyl groups, straight-chain alkyl groups substituted with C3-C8 cycloalkyl groups, or unsubstituted C8-C8 cycloalkyl groups 24 Cycloalkyl.

[0020] In this invention, X is a residue portion of a compound (amino acid) containing amino and carboxyl groups after substitution, which is connected to a hydrophobic segment via an ester bond and to Y via an amide bond. Preferably, X is an amino acid residue.

[0021] In this invention, the amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, sarcosine, histidine, selenocysteine, and pyrrolidone, etc.

[0022] In this invention, amino acid residues are conventionally referred to as the groups remaining after the removal of the carboxyl group and / or the hydrogen atom from the amino group of an amino acid, i.e., the residue portion after substitution.

[0023] In this 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, etc.

[0024] Preferably, Y contains an ethylene glycol segment, and also contains an amide group and / or amino acid residues. Y is a straight chain or a straight-chain structure; preferably, Y is a straight chain containing a carbonyl group.

[0025] In this 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 2 to 20, more preferably 3 to 18, and even more preferably 4 to 16.

[0026] In this invention, Y is connected to maleimide via an amide bond. Preferably, the amide bond and the maleimide group are connected by a substituted or unsubstituted alkyl group. Preferably, the alkyl group contains 1 to 20 carbon atoms, more preferably, it contains 2 to 15 carbon atoms, and even more preferably, it 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 number within the range.

[0027] In this invention, the raw materials for preparing lipid nanoparticles include not only the lipid compounds mentioned above, but also other raw materials. These other raw materials are other conventional lipid nanoparticle raw materials that 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.

[0028] This invention discloses a method for preparing albumin-modified lipid nanoparticles. The method involves preparing lipid nanoparticles using lipid nanoparticle raw materials, and then modifying the lipid nanoparticles with albumin to obtain albumin-modified lipid nanoparticles. Specifically, lipid nanoparticles are prepared using lipid nanoparticle raw materials comprising the aforementioned lipid compounds, and then albumin is modified onto the lipid nanoparticles to obtain albumin-modified lipid nanoparticles. The lipid nanoparticle raw materials comprising the aforementioned lipid compounds include, in addition to the aforementioned lipid compounds, other conventional lipid nanoparticle raw materials, such as ionizable cationic lipids, cholesterol, phospholipid derivatives, and polyethylene glycol lipids.

[0029] This invention discloses a method for preparing albumin-modified drug-loaded lipid nanoparticles. The method involves preparing drug-loaded lipid nanoparticles using lipid nanoparticle raw materials and a drug as raw materials, and then modifying the drug-loaded lipid nanoparticles with albumin to obtain albumin-modified drug-loaded lipid nanoparticles. Specifically, lipid nanoparticles are prepared using lipid nanoparticle raw materials including the aforementioned lipid compound and a drug as raw materials, and then albumin is modified onto the lipid nanoparticles to obtain albumin-modified lipid nanoparticles. The lipid nanoparticle raw materials including the aforementioned lipid compound include, in addition to the aforementioned lipid compound, other conventional lipid nanoparticle raw materials, such as ionizable cationic lipids, cholesterol, phospholipid derivatives, polyethylene glycol lipids, etc.

[0030] As an example, the preparation method of drug-loaded lipid nanoparticles includes the following steps: nucleic acid-containing aqueous phase and lipid organic phase are mixed using a physical mixing method or a microfluidic method to prepare nucleic acid-loaded lipid nanoparticles. The specific preparation of the aqueous and organic phases follows conventional techniques, and water and alcohol can be used as solvents, respectively.

[0031] In this invention, the lipid compound accounts for 1% to 35% of the other raw materials, preferably 1.5% to 20%, more preferably 1.5% to 15%, even more preferably 2% to 10%, and even more preferably 3% to 6%.

[0032] In this invention, the drug includes one or more of nucleic acids, such as mRNA, circular RNA, siRNA, microRNA, and antisense nucleic acids.

[0033] In this invention, albumin contains sulfhydryl groups, such as human serum albumin and bovine serum albumin.

[0034] In this invention, lipid nanoparticles are mixed and incubated with albumin to obtain albumin-modified lipid nanoparticles; drug-loaded lipid nanoparticles are mixed and incubated with albumin to obtain albumin-modified drug-loaded lipid nanoparticles.

[0035] In this invention, the incubation temperature is 0℃ to room temperature; preferably, lipid nanoparticles are mixed with albumin and incubated at room temperature, and then left to stand at 0℃ to 5℃ to obtain albumin-modified lipid nanoparticles; drug-loaded lipid nanoparticles are mixed with albumin and incubated at room temperature, and then left to stand at 0℃ to 5℃ to obtain albumin-modified drug-loaded lipid nanoparticles.

[0036] The drug-loaded liposome nanoparticles of this invention are a nucleic acid (mRNA) delivery system for lymph node targeting by albumin-modified site-directed modification. At low pH values, they facilitate mRNA endosome escape and enhance translational function. At physiological pH values, they are neutral, significantly reducing potential safety and toxicity. Simultaneously, they can reduce antibody binding to serum proteins and clearance by phagocytes, prolonging systemic circulation time and minimizing the risk of allergic reactions. They also have stabilizing and membrane-fusion-promoting effects. The nanoparticles of this invention possess natural lymph node targeting capabilities, significantly enhancing the uptake capacity of antigen-presenting cells, thereby efficiently activating the body's immune response. Furthermore, LNPs are easy to design and prepare, enabling the creation of both effective and safe vaccines, thus showing promising application prospects.

[0037] This invention discloses the application of the above-mentioned lipid compounds, albumin-modified lipid nanoparticles, or albumin-modified drug-loaded lipid nanoparticles in the preparation of biological products or drugs.

[0038] This invention discloses the application of the above-mentioned lipid compounds, albumin-modified lipid nanoparticles, or albumin-modified drug-loaded lipid nanoparticles in the preparation of antitumor drug reagents.

[0039] A biological product whose active ingredient includes the aforementioned albumin-modified drug-loaded lipid nanoparticles.

[0040] In this 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 vaccine, AIDS vaccine, viral pneumonia vaccine, tuberculosis vaccine, respiratory syncytial virus vaccine, enterovirus vaccine, intestinal mucosa-associated tumor vaccine or tumor vaccine, etc.

[0041] Preferably, the biological product is used for the prevention and / or treatment of infectious diseases or tumors; tumors include solid tumors or hematologic tumors, such as melanoma, lung cancer, colon cancer and other types of tumors.

[0042] Furthermore, the present invention discloses an antitumor drug whose active ingredient includes the above-mentioned albumin-modified drug-loaded lipid nanoparticles.

[0043] In this invention, the biological product or drug can be applied by methods such as injection, oral administration, or topical application.

[0044] This invention uses the above-mentioned albumin-modified drug-loaded lipid nanoparticles as the sole active ingredient of the antitumor drug, or the above-mentioned albumin-modified drug-loaded lipid nanoparticles can be used in combination with other antitumor drugs for treatment.

[0045] This invention prepares lipid nanoparticles capable of carrying nucleic acids by combining a lipid compound (functionalized low molecular weight PEG-maleimide derivative) with commonly used nucleic acid delivery lipid raw materials; nucleic acids are added to the lipid nanoparticle preparation raw materials to prepare nucleic acid-loaded lipid nanoparticles; then, through a Michael addition reaction, the maleimide on the surface of the nanoparticles is chemically coupled to the unique thiol group of albumin, thereby obtaining nucleic acid-loaded lipid nanoparticles mRNA-LNP with surface-specifically coupled albumin.

[0046] This invention provides albumin-modified lipid nanoparticles, their preparation method, and their application in mRNA delivery and anti-tumor activity. The advantages of the albumin-modified nanoparticles are as follows: 1) Maleimide on the nanoparticle surface chemically couples with the unique thiol group of albumin, achieving albumin-modified nanoparticle surface modification. This counteracts the steric hindrance caused by PEG chain antibodies, reducing the immunogenicity of the nanoparticles to the body; 2) Utilizing the lymph node targeting ability of the surface-coupled albumin, the lymph node enrichment of the nanoparticles can be improved, allowing them to exert their effects more effectively. Attached Figure Description

[0047] Figure 1 Lipid nanoparticles that are site-modified from albumin.

[0048] Figure 2 This is a schematic diagram illustrating the preparation of the intermediate DMG-G.

[0049] Figure 3 This is a schematic diagram illustrating the preparation of the intermediate Mal-PEG4-OH.

[0050] Figure 4 The intermediates Mal-PEG8-OH and Mal-PEG 12 -OH, Mal-PEG 16 A schematic diagram of the preparation of -OH.

[0051] Figure 5 This is a schematic diagram illustrating the preparation of lipid compounds.

[0052] Figure 6 Fluorescence distribution of nanoparticle-delivered drugs in lymph nodes: A. Lymph nodes captured by a small animal imaging system; B. Fluorescence bar graph.

[0053] Figure 7The data represent the tumor growth curves of mice bearing B16-OVA tumors treated with nanoparticles. Data were processed according to standard statistical methods. ns indicates no statistical difference, * indicates P<0.05, and **P<0.01. Detailed Implementation

[0054] This invention belongs to the field of biomedical technology, specifically relating to a functionalized low molecular weight PEG-maleimide lipid compound. It is used together with commonly used nucleic acid delivery lipid composition materials to prepare nucleic acid-loaded lipid nanoparticles. Through Michael addition reaction, the maleimide on the surface of the nanoparticles is chemically coupled with the unique thiol group of albumin, thereby obtaining nanoparticles with albumin site-specific modification on the surface of the lipid nanoparticles.

[0055] To address the limitations of existing mRNA-loaded lipid nanoparticles in targeting lymph nodes and the immunogenicity issues arising from their surface PEG chains, this invention provides a functionalized low-molecular-weight PEG-maleimide derivative and its preparation method. This derivative is combined with commonly used nucleic acid delivery lipid compositions to prepare nucleic acid-loaded lipid nanoparticles. Through a Michael addition reaction, the maleimide on the nanoparticle surface is chemically coupled to the unique thiol group of albumin, thereby obtaining nanoparticles with albumin-modified surfaces. See details... Figure 1 .

[0056] The albumin-modified lipid nanoparticles disclosed in this invention are prepared by site-specific modification of lipid nanoparticles with albumin, and drug-loaded lipid nanoparticles are prepared by site-specific modification of drug-loaded lipid nanoparticles with albumin. The lipid nanoparticles are prepared using a functionalized low-molecular-weight PEG-maleimide derivative as the lipid compound and existing conventional lipid raw materials. The drug loaded includes nucleic acids. Existing conventional lipid raw materials are ionizable cationic lipids, polyethylene glycol-modified lipids, cholesterol, and phospholipid derivatives. The carrier of this invention includes a functionalized low-molecular-weight PEG lipid compound and commonly used nucleic acid delivery composition materials: ionizable cationic lipids, polyethylene glycol-modified lipids, cholesterol, and phospholipid derivatives; the nucleic acids are selected from one or more of mRNA, circular RNA, siRNA, microRNA, and antisense nucleic acids.

[0057] In this invention, the lipid compound is a functionalized low molecular weight PEG-maleimide derivative, and its structure is as shown in formula (1):

[0058] (1)

[0059] Where R1 and R2 are -C(=O)OR, and R is the unsubstituted C8-C. 24 Straight-chain alkyl, unsubstituted C6-C 18Branched alkyl groups, straight-chain alkyl groups substituted with C3-C8 cycloalkyl groups, or unsubstituted C8-C8 cycloalkyl groups 24 cycloalkyl;

[0060] X represents an amino acid residue;

[0061] Y contains ethylene glycol segments, as well as amide groups and / or amino acid residues;

[0062] Mal is a maleimide group;

[0063] n is 1 to 3.

[0064] In this invention, the functionalized low molecular weight PEG lipid compound accounts for 1% to 35% (mol / mol) of existing conventional lipid raw materials; the functionalized low molecular weight PEG lipid compound is selected from one or more of maleimide-containing lipid compounds and maleimide derivatives, such as maleimide lipid compounds. Because it can undergo a Michael addition reaction with the unique thiol group of albumin, it can perform site-specific modification on the surface of lipid nanoparticles. Examples include Mal-PEG4-G-DMG, Mal-PEG8-G-DMG, and Mal-PEG. 12 -G-DMG, Mal-PEG 16 One or more of the following: -G-DMG, Mal-PEG4-VC-PAB-G-DMG, and (Mal-PEG4)2-Lys-DMG.

[0065] In this invention: ionizable cationic lipids, including but not limited to one or more of SM-102, ALC-0315, DLin-MC3-DMA, cKK-E12, L319, DLin-KC2-DMA, C12-200, DOTAP, and 306Oi10, account for 30% to 70% (mol / mol) of the conventional lipid raw materials, preferably 40% to 60% (mol / mol);

[0066] 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) of existing conventional lipid raw materials, preferably 5% to 15% (mol / mol).

[0067] Cholesterol (including its derivatives) includes, but is not limited to, one or more of β-sitosterol, cholesterol, cholesterol ketone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol, which account for 20% to 45% (mol / mol) of existing conventional lipid raw materials, preferably 30% to 40% (mol / mol).

[0068] Polyethylene glycol-modified 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) of existing conventional lipid raw materials, preferably 0.8% to 10% (mol / mol).

[0069] The molar amount of existing conventional lipid raw materials is the sum of the molar amounts of ionizable cationic lipids, phospholipid derivatives, cholesterol, and polyethylene glycol-modified lipids.

[0070] In this invention, the mass ratio of ionizable cationic lipids to nucleic acids is (1-50):1; preferably (5-30):1; more preferably (10-20):1.

[0071] In this invention, the hydrated particle size of the lipid nanoparticles is 10–120 nm; preferably 80–100 nm.

[0072] In this invention, the encapsulation rate of nucleic acids in the drug-loaded lipid nanoparticles is 30%–99%; preferably 80%–99%.

[0073] In this invention, the method for preparing albumin-modified lipid nanoparticles includes the following steps: preparing lipid nanoparticles by mixing an aqueous phase and a lipid organic phase through a physical mixing method or a microfluidic method.

[0074] In this invention, the preparation method of albumin-modified drug-loaded lipid nanoparticles includes the following steps: preparing nucleic acid-loaded lipid nanoparticles by mixing an aqueous phase containing nucleic acids with a lipid organic phase through a physical mixing method or a microfluidic method.

[0075] The aqueous phase includes water-soluble drugs and buffer solutions; the lipid organic phase includes lipid compounds, as well as conventional ionizable cationic lipids, phospholipid derivatives, cholesterol, polyethylene glycol-modified lipids, and alcohol solvents.

[0076] Specifically, water-soluble drugs, such as nucleic acids (mRNA), are dissolved in a buffer solution as the aqueous phase. As an example, water-soluble drugs, such as nucleic acids (mRNA), are dissolved in a citrate buffer solution to a final concentration of 10–300 μg / mL, preferably 30–250 μg / mL.

[0077] Specifically, the organic phase uses various organic solvents such as small molecule alcohol solvents (e.g., ethanol) to dissolve the carrier material (functionalized low molecular weight PEG lipid compounds, and commonly used ionizable cationic lipids: phospholipid derivatives: cholesterol: polyethylene glycol-modified lipids, with the molar ratio of the latter four raw materials being 50:10:38.5:1.5); among which, low molecular weight PEG lipid compounds account for 1.5% to 6% of the latter four raw materials, by molar weight.

[0078] In this invention, cationic lipids can be ionized under acidic conditions, protonated to form positively charged lipids, which then bind to negatively charged nucleic acids via electrostatic interactions to form nucleic acid-loaded nanoparticles.

[0079] As an example, the physical mixing method includes the following steps: taking an aqueous phase containing nucleic acid and adding it to a lipid organic phase in a vortex state, mixing well and letting it stand to obtain the nucleic acid-loaded lipid nanoparticles.

[0080] Optionally, the product obtained by the physical mixing method may be dialyzed, for example, in a PBS buffer solution (11.8 mM, pH 7.4) of more than 1000 times its volume for more than 4 hours.

[0081] The microfluidic method includes the following steps: using a syringe to draw up a lipid organic phase (lipid concentration: 3-15 mg / mL) and an aqueous phase containing (50-200 μg / mL) nucleic acid, respectively, and injecting the lipid organic phase and the aqueous phase into the microfluidic chip at a flow rate of 1:3 to mix them. After the aqueous phase solution in the syringe is exhausted, the liquid collection is stopped to obtain lipid nanoparticles loaded with nucleic acid; preferably, the microfluidic method also includes dialyzing the obtained mixture, for example, dialyzing in PBS (11.8 mM, pH=7.4) at a volume greater than 1000 times its volume for more than 4 hours.

[0082] The lipid nanoparticles or drug-loaded lipid nanoparticles prepared in this invention are mixed with an albumin solution and incubated to obtain lipid nanoparticles or drug-loaded lipid nanoparticles with albumin conjugated on the surface. Specifically, the lipid nanoparticles or drug-loaded lipid nanoparticles are mixed with an albumin solution and incubated at room temperature, then placed at 4°C to obtain lipid nanoparticles or drug-loaded lipid nanoparticles with albumin conjugated on the surface. Preferably, they are placed at 4°C for 10–50 hours, more preferably for 12–30 hours. As an example, the lipid nanoparticles or drug-loaded lipid nanoparticles are mixed with an albumin solution and incubated at room temperature, then placed at 4°C for 10–30 hours, and then subjected to agarose gel column chromatography using PBS (11.8 mM, pH=7.4) as the mobile phase to obtain lipid nanoparticles with albumin conjugated on the surface carrying nucleic acid mRNA-LNP. The steric hindrance introduced by the surface conjugation of albumin can reduce or overcome the immunogenicity of existing nanoparticles coated with PEG.

[0083] This invention discloses the application of the above-mentioned mRNA-LNP containing functionalized low molecular weight PEG lipid compounds as a biological product. The biological product is a vaccine, preferably an mRNA vaccine. This includes, but is not limited to: influenza vaccines, HIV vaccines, viral pneumonia vaccines, tuberculosis vaccines, respiratory syncytial virus vaccines, enterovirus vaccines, intestinal mucosa-associated tumor vaccines, or lung cancer vaccines, etc.

[0084] The biological products are used for the prevention and / or treatment of infectious diseases or tumors, including but not limited to melanoma, colon cancer, and other types of tumors. The biological products can be applied via injection, oral administration, or topical application.

[0085] This invention provides a lipid compound, which is a functionalized low molecular weight PEG-maleimide derivative, and its exemplary chemical structure is shown in formula (2) below:

[0086] (2)

[0087] Wherein, X is a polypeptide amino acid chain containing different types of amino acid residues and PEG fragments.

[0088] In a preferred embodiment of the present invention, the lipid compound contains PEG-functionalized linker arms of different chain lengths, namely: MC-PEG4-DMG, MC-PEG8-DMG, and MC-PEG. 12 -DMG, MC-PEG 16 -DMG, MC-PEG4-VC-PNP-G-DMG, MC-PEG4-Lys(MC-PEG4)-DMG; their chemical structural formulas are respectively (3) to (8), as follows:

[0089] (3)

[0090] (4)

[0091] (5)

[0092] (6)

[0093] (7)

[0094] (8)

[0095] Unless otherwise specified, MC-PEG was selected for the experiments in this invention. 16 -DMG (chemical formula 6) lipid compounds have outstanding biological effects in in vitro cell transfection and in vivo lymphatic distribution and transfection studies.

[0096] The above-mentioned functionalized low molecular weight PEG-maleimide derivatives were combined with commonly used nucleic acid delivery lipid composition materials and nucleic acid drugs to prepare nucleic acid-loaded lipid nanoparticles. Through Michael addition reaction, the maleimide on the surface of the nanoparticles was chemically coupled with the unique thiol group of albumin, thereby obtaining nucleic acid-loaded lipid nanoparticles mRNA-LNP with surface-specifically coupled albumin.

[0097] The following specific embodiments 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, the reaction is monitored by TLC using G-type silica gel plates, with petroleum ether:ethyl acetate = 10:1 and ultraviolet color development; the column chromatography packing is 200-mesh H-type silica gel.

[0098] The animal experiments met the relevant requirements of Soochow University.

[0099] Nucleic acid information: EGFP mRNA (encoding enhanced green fluorescent protein), Luc mRNA (encoding luciferase), and OVA mRNA (encoding ovalbumin) are commonly used functional mRNAs and are commercially available. Albumin is human serum albumin; dichloropolymer resin was purchased from Nankai Hecheng Technology Co., Ltd.

[0100] I. Synthesis of functionalized low molecular weight PEG-maleimide derivatives (lipid compounds)

[0101] See Figure 2 Intermediate B: 4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane was synthesized using commercially available product A as the starting material; Intermediate C: 3-benzyloxy-1,2-propanediol was synthesized using intermediate B as the starting material; Intermediate D: 3-benzyloxy-1,2-propanediol tetracosanoate was synthesized using intermediate C as the starting material; Intermediate DG: 1,2-dimyristic acid glyceride was synthesized using intermediate DG and commercially available product Fmoc-Gly-OH as the starting material; Intermediate E: 3-(9-fluorenylmethoxycarbonylglycyloxy)propane-1,2-dimethyltetracosanoate was synthesized using intermediate E as the starting material; Intermediate DMD-G: 1,2-tetradecanoylglycerol-3-glycine was synthesized using intermediate E as the starting material.

[0102] See Figure 3 and Figure 4 Using Fmoc-PEG4-OH and 6-maleimide hexanoic acid as raw materials, intermediates Mal-PEG4-OH, Mal-PEG8-OH, and Mal-PEG were synthesized, respectively. 12 -OH, Mal-PEG 16 -OH, all the intermediates synthesized above can be uniformly expressed as Mal-PEG. n -OH.

[0103] See Figure 5 Using the intermediate Mal-PEG n The lipid compound Mal-PEGn-DMG was synthesized from -OH and intermediate DMG-G.

[0104] A lipid compound (functionalized low molecular weight PEG-maleimide derivative, Mal-PEGn-DMG) and a commonly used nucleic acid delivery lipid composition (ionizable cationic lipid: phospholipid derivative: cholesterol: polyethylene glycol-modified lipid, with a molar ratio of 50:10:38.5:1.5) were dissolved together in an organic solvent such as ethanol to form the organic phase. Nucleic acid (mRNA) was dissolved in a citrate buffer solution to form the aqueous phase. The above lipid organic phase and the nucleic acid-containing aqueous phase were mixed by physical mixing or microfluidic methods to prepare the nucleic acid-loaded lipid nanoparticles mRNA-LNP. Then, the nanoparticles were mixed with an albumin solution and incubated sequentially at room temperature and overnight at 4°C to allow the thiol groups on the albumin to couple with the maleimide on the nanoparticle surface. The nanoparticles were then purified by agarose gel column chromatography to obtain the nucleic acid-loaded lipid nanoparticles HSA-mRNA-LNP with surface-specifically coupled albumin.

[0105] Example 1

[0106] 1. Preparation of intermediate B: 4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane

[0107] 515 mg of NaH was weighed into a double-necked flask. 10 mL of DMF was added under a nitrogen atmosphere and normal pressure. In an ice-water bath, commercially available glycerol acetal (compound A, 1.0 g) was added. After regular stirring, α-bromomethylbenzene was added, and the mixture was stirred at room temperature. After the reaction was complete as monitored by TLC, 10 mL of ice water was added to quench the reaction. The mixture was extracted with diethyl ether (60 mL × 3), and the organic phases were combined and washed three times with saturated NaCl water. The organic phase was concentrated under reduced pressure and separated by column chromatography to obtain 1.3 g of compound B (yield 77.33%). Proton NMR spectroscopy identification: 1 1H 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). These 1H NMR data confirm that the obtained compound is correct.

[0108] 2. Preparation of intermediate C: 3-benzyloxy-1,2-propanediol

[0109] Intermediate B (800 mg) was weighed into a single-necked flask, dissolved in 14 mL of acetic acid and 6 mL of water, and stirred at 65 °C for 1 hour. The mixture was then allowed to cool naturally to room temperature. A saturated NaHCO3 aqueous solution was added to the reaction system to adjust the pH to 7. The mixture was then extracted with dichloromethane (60 mL × 3). The organic phases were combined, dried, concentrated, and purified by column chromatography to give 500 mg of compound C (yield 76.33%). Proton NMR spectroscopy was performed. 1 1H 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). These 1H NMR data confirm that the obtained compound is correct.

[0110] 3. Preparation of intermediate D: 3-benzyloxy-1,2-propanediol tetracosanoate

[0111] Intermediate C (500 mg), N,N'-dicyclohexylcarbodiimide (1700 mg), and 4-dimethylaminopyridine (56.5 mg) were weighed into a 50 mL single-necked flask. 20 mL of dichloromethane was added to dissolve the compounds. After stirring at room temperature, tetradecanoic acid (1880 mg) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete as monitored by TLC, the mixture was filtered to remove solid impurities. An equal volume of water was added to the filtrate, and the mixture was washed three times with dichloromethane. The organic phases were combined, dried, concentrated, and purified by column chromatography to give 1300 mg of compound D (yield 78.64%). Proton NMR spectroscopy was performed. 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 proton NMR data confirm that the obtained compound is correct.

[0112] 4. Preparation of intermediate DMG: 1,2-dimyristic acid glyceride

[0113] Intermediate D (500 mg) was weighed into a 50 mL single-necked flask, dissolved in acetic acid / ethanol (6 mL:3 mL, 2:1), followed by the addition of Pd / C (400 mg, 10% purity). The mixture was purged three times with hydrogen gas and stirred at room temperature for 1 hour under a hydrogen atmosphere. The reaction solution was diluted with dichloromethane, the palladium catalyst on carbon was filtered off, and the soluble matter in the solid was dissolved in dichloromethane. The combined organic phase dichloromethane solutions were washed once with saturated NaHCO3 aqueous solution and three times with saturated NaCl water. After drying and concentration, the solution was subjected to silica gel column chromatography to obtain 350 mg of compound DMG (yield 82.35%). Hydrogen spectroscopy identification: 1 1H 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, 7.5 Hz, 3H), 1.27 (d, J = 10.9 Hz, 41H), 0.88 (t, J = 6.9 Hz, 6H). These 1H NMR data confirm that the obtained compound is correct.

[0114] 5. Preparation of intermediate E: 3-(9-fluorenylmethoxycarbonylglycyloxy)propane-1,2-dimethyltetracosanoate

[0115] Weigh 200 mg of intermediate DMG, 176 mg of commercially available Fmoc-Gly-OH, and 4-dimethylaminopyridine (48 mg) into a 25 mL single-necked flask. Dissolve in 10 mL of dichloromethane, and place the reaction system at 0 °C. Add 120 mg of N,N-dicyclohexylcarbodiimide. After the reaction is complete as monitored by TLC, filter out solid impurities. Add an equal volume of water to the filtrate and wash three times with dichloromethane. Combine the organic phases, dry and concentrate, and purify by column chromatography to obtain 253 mg of compound E (yield 82.14%). Identification by 1H NMR: 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 1H NMR data confirm that the obtained compound is correct.

[0116] 6. Preparation of intermediate DMG-G: 1,2-Tetradecanoylglycerol-3-glycine

[0117] Compound E (50 mg) was weighed into a 25 mL single-necked flask, dissolved in 20 mL of dichloromethane, and the reaction system was placed at 0 °C. 11 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added, and the mixture was stirred for 0.5 hours. Then, an equal volume of water was added to dilute the reaction solution. The mixture was extracted with ethyl acetate (10 mL × 3), dried, concentrated, and purified by silica gel column chromatography to obtain 21 mg of compound DMG-G (yield 58.39%). Proton NMR spectroscopy was performed. 1 1H 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). These 1H NMR data confirm that the obtained compound is correct.

[0118] Example 2

[0119] 1. Using Fmoc-PEG4-OH and 6-maleimide hexanoic acid as raw materials, intermediates Mal-PEG4-OH, Mal-PEG8-OH, and Mal-PEG were synthesized, respectively. 12 -OH, Mal-PEG 16 -OH. With MC-PEG 16 -The preparation of DMG is used as an example.

[0120] Weigh 1.0 g of dichloromethane resin and add it to the solid-phase reaction column. After washing twice with dichloromethane, swell the resin with dichloromethane for 30 min and then remove the solvent.

[0121] The first Fmoc-PEG4-OH (1.6 g) was dissolved in dichloromethane, and 0.6 mL of N,N-diisopropylethylamine was added for activation. This solution was added to the solid-phase reaction tube containing the swollen resin. The reaction was stirred for 3 hours under nitrogen protection, and then 30 mL of MeOH was added and the mixture was sealed for 1 hour. After washing 4 times with DMF, the MeOH shrank and was dried under vacuum. 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 deprotection, the mixture was washed 5 times each with DMF and dichloromethane to obtain NH2-PEG4-dichloro resin.

[0122] The second amino acid analog Fmoc-PEG4-OH (1.6 g) and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL of DMF. N,N-diisopropylcarbodiimide (171 μL) was added under ice bath conditions to activate the solution for 30 min. The solution was then added to a solid-phase reaction tube containing the resin from the previous step. Dimethylaminopyridine (27 mg) was added and the mixture was stirred for 3 hours under nitrogen protection. 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 deprotection, the solution was washed 5 times each with DMF and dichloromethane to obtain NH2-PEG4-PEG4-dichloro resin.

[0123] The third amino acid analog Fmoc-PEG4-OH (1.6 g) and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL of DMF. N,N'-diisopropylcarbodiimide (171 μL) was added under ice bath conditions to activate the solution for 30 min. The solution was then added to a solid-phase reaction tube containing the resin from the previous step. Dimethylaminopyridine (27 mg) was added and the mixture was stirred for 3 hours under nitrogen protection. 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 deprotection, the solution was washed 5 times each with DMF and dichloromethane to obtain NH2-PEG4-PEG4-PEG4-dichloro resin.

[0124] The fourth amino acid analog, Fmoc-PEG4-OH (1.6 g), and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL of DMF. N,N-diisopropylcarbodiimide (171 μL) was added under ice bath conditions to activate the solution for 30 min. The solution was then added to a solid-phase reaction tube containing the resin from the previous step. Dimethylaminopyridine (27 mg) was added and the mixture was stirred for 3 hours under nitrogen protection. 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 deprotection, the solution was washed 5 times each with DMF and dichloromethane to obtain NH2-PEG4-PEG4-PEG4-PEG4-dichloro resin.

[0125] 6-maleimide hexanoic acid (234 mg) and 1-hydroxybenzotriazole (150 mg) were dissolved in 20 mL of DMF. N,N'-diisopropylcarbodiimide (171 μL) was added under ice bath conditions for 30 min to activate the solution. This solution was then added to a solid-phase reaction column containing the resin from the previous step. Dimethylaminopyridine (27 mg) was added, and the reaction was stirred for 3 hours under nitrogen protection to obtain Mal-PEG. 16 -OH.

[0126] Mal-PEG 16 -OH removal from the resin: The above resin was vacuum dried and transferred to a round-bottom flask. It was removed with a trifluoroethanol (TFE)-dichloromethane mixture (1:4, v / v) for 2 hours, then filtered. When the filtrate was concentrated to one-quarter of its original volume, the concentrate was added to 10 times the volume of diethyl ether for precipitation. The mixture was allowed to stand at 4°C for 2 hours, then filtered again. The filter cake was washed 6 times with diethyl ether and vacuum dried to obtain crude polypeptide powder.

[0127] Separation and purification: The crude peptide was separated and purified using high-performance liquid chromatography (HPLC). The conditions were: phase A was 0.1% trifluoroacetic acid aqueous phase, phase B was acetonitrile phase, phase A-phase = 10%-90%, and the chromatography time was 20 min. The resulting peptide sample (purity ≥ 95%) was 253 mg Mal-PEG. 16 -OH. Mass spectrometry identification: MS-ESI (m / z): [MH]- calcd for: 1128.65; found: 1127.79. The above mass spectrometry data confirm that the obtained compound is correct.

[0128] Following the method described above, Mal-PEG4-OH, Mal-PEG8-OH, and Mal-PEG4-OH were obtained by using one Fmoc-PEG4-OH, two Fmoc-PEG4-OH, and three Fmoc-PEG4-OH, respectively. 12 -OH.

[0129] 2. Using the intermediate Mal-PEG 16 Using -OH and intermediate DMG-G as raw materials, Mal-PEG is synthesized. 16 -DMG.

[0130] Weigh out compound DMG-G (40 mg) and Mal-PEG. 16 -OH (43 mg) and 2-(7-azobenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (40 mg) were dissolved in 10 mL of DMF in a 25 mL single-necked flask. The reaction system was then placed at 0 °C, and N,N-diisopropylethylamine (25 μL) was added. After the reaction was monitored by TLC until complete, an equal volume of water was added to the reaction system, and the mixture was washed three times with dichloromethane. The organic phases were combined, dried, concentrated, and purified by silica gel column chromatography to obtain 55 mg of Mal-PEG. 16 -DMG (yield 44%). Mass spectrometry identification: MS-ESI (m / z): [M+2H] 2+ Calculation result for: 1751.11; Found value: 876.56. The above mass spectrometry data confirms that the obtained compound is correct.

[0131] Following the method described above, the intermediate Mal-PEG was used. n Using -OH and intermediate DMG-G as raw materials, Mal-PEGn-DMG was synthesized, and mass spectrometry data confirmed that the obtained compound was correct.

[0132] Example 3

[0133] 1. Preparation and characterization of nucleic acid-loaded lipid nanoparticles

[0134] Ionizable cationic lipids can be protonated under acidic conditions to form positively charged lipids, which then bind to negatively charged nucleic acids via electrostatic interactions, forming nucleic acid-loaded lipid nanoparticles (mRNA-LNPs). The mRNA-LNPs prepared in this invention have an ionizable cationic lipid to encapsulated nucleic acid mass ratio of (10–30):1, and a molar ratio of ionizable cationic lipid:helper phospholipid:cholesterol:polyethylene glycol lipid of 50:10:38.5:1.5. The ionizable cationic lipid is SM-102.

[0135] Note: The chemical name of SM-102 is: 8-[(2-hydroxyethyl)(6-oxo-6-decoxyhexyl)amino]octanoic acid (heptadecane-9-yl) ester; cholesterol; the auxiliary phospholipid is DSPC; polyethylene glycol lipid DMG-PEG2000; the prepared product is a 4-component nucleic acid lipid nanoparticle mRNA-LNP.

[0136] The five-component lipid nanoparticles HSA-mRNA-LNP of the present invention are prepared by adding Mal-PEG4-DMG (in proportions of 1.5%, 3.0%, 6.0%, and 12%, respectively) to the above four components. The proportion of Mal-PEG4-DMG added to HSA-mRNA-LNP is based on the relative molar ratio to the four lipid components.

[0137] Preparation method: mRNA was dissolved in 50 mM sodium citrate buffer (pH 5.0) to a final concentration of 200 μg / mL (aqueous phase). SM-102:cholesterol:DSPC:DMG-PEG2000 were mixed in a molar ratio of 50:38.5:10:1.5 to form a lipid mixture (oil phase). A certain molar proportion of Mal-PEG was then added to this four-component mixture. 16 -DMG lipids (1.5%, 3.0%, 6.0%, 12%) were used to form five-component liposomes, each dissolved in ethanol to form the organic phase. The flow rates of the aqueous and oil phases were controlled via microfluidics to mix the mRNA and lipid mixture at a 3:1 volume ratio. The buffer environment was replaced with pH 7.4 PBS using dialysis or tangential flow to remove ethanol, resulting in four mRNA-LNPs: PEG... 16 -LNP (1.5%) PEG 16 -LNP (3.0%) PEG 16 -LNP (6.0%) PEG 16 -LNP (12%) .

[0138] The product of only four components is Moderna-LNP.

[0139] Following the method described above, nucleic acid-loaded nanoparticles PEGn-LNP were prepared using Mal-PEGn-DMG.

[0140] 2. Preparation and characterization of albumin-modified nucleic acid-loaded lipid nanoparticles

[0141] Albumin solutions of 10 mg / mL (40 μL, 80 μL, 160 μL, and 320 μL) were added to PEGn-LNPs prepared with different PEG units from Mal-PEGn-DMG. After incubation at room temperature for 2 hours, the solutions were placed at 4°C for 20 hours. The solutions were then purified using an agarose gel filtration column with PBS (11.8 mM, pH 7.4) as the mobile phase to obtain a solution of five components of albumin-conjugated nucleic acid-loaded lipid nanoparticles (HSA-mRNA-LNPs). These solutions were then concentrated using 100K Millipore ultrafiltration centrifuge tubes to obtain HSA-mRNA-LNPs modified with albumin and containing different PEG units at varying concentrations. Due to the large amount of information required for each nanoparticle, abbreviations are used. For example: Mal-PEGn-DMG. 16 - Nanoparticles containing 1.5% DMG can be abbreviated as HSA-PEG. 16 -LNP （1.5%） .

[0142] The final nanoparticle products prepared with different formulations were diluted 10 times with diluent and added to 1 mL of the particle size cell. The cells were then placed on a Malvern ZetaSizer instrument to detect the characterization data of hydrated particle size, polydispersity index, and surface potential. See Table 1 for details. The mRNA was EGFP mRNA, the mass ratio of ionizable lipids to mRNA was 20:1, and the albumin solution was 160 μL.

[0143] The nucleic acid encapsulation efficiency of each nanoparticle was determined using the Ribogreen kit and 1% Triton as a demulsifier, and the results were 90-94%.

[0144] Table 1. Hydrated particle size, polydispersity index, and surface potential of nanoparticles

[0145]

[0146] The surface potential column in Table 1 shows: Moderna-LNP with four components is -1.0 mV; PEG with five components... 16 -LNP is -1.4mV; 5-component HSA-PEG 16 -LNP has a potential of -5.7 mV; free human serum albumin (HSA) has a potential of -13 mV. Therefore, 1) HSA has a large negative charge, and the five-component PEG... 16 -LNP has a very small negative charge, while HSA-PEG 16The increased negative charge of -LNP interacts with the fifth component, Mal-PEG, in the nanoparticles. 16 - The addition of DMG and coupling with HSA; 2) HSA-PEG 16 The albumin site-coupled on the LNP surface forms a spatial shielding effect on the PEG on its surface. Therefore, it can promote the reduction of PEG immunogenicity on the nanoparticle surface, which is one of the significant technical advancements achieved by this invention.

[0147] Example 4

[0148] 1. In vitro cell transfection capability of nanoparticles

[0149] Following the method described above, EGFP mRNA (mRNA expressing green fluorescent protein) was used as the encapsulated nucleic acid to prepare HSA-PEGn-LNP loaded with EGFP mRNA at a mass ratio of 20:1, and the albumin solution was 160 μL. The experimental group was set up as: HSA-PEGn-LNP. n -LNP (1.5%) HSA-PEG n -LNP (3.0%) HSA-PEG n -LNP (6.0%), HSA-PEG n -LNP (12%) The control group consisted of Moderna-LNP (four-component nanoparticles, comparable to commercially available products), and the same experimental procedures were performed to evaluate the cell transfection ability of the lipid nanoparticles. One day before the experiment, DC2.4 cells were seeded into 48-well cell culture plates, with 50,000 cells per well. When the cell confluence reached 70%-80%, the nanoparticles from each group were added, gently shaken to mix, and incubated at 37°C and 5% CO2 for 3 hours. Cells were digested with trypsin to obtain single-cell suspensions, and the mean fluorescence intensity of the cells was detected using flow cytometry to verify the in vitro cell transfection efficiency of each group of nanoparticles. The results are detailed in Table 2.

[0150] Table 2 Cell transfection capability of nanoparticles

[0151]

[0152] Transfection status of DC2.4 cells can be observed: experimental group HSA-PEG 16 -LNP (fluorescence value 2746), control group PEG 16 -LNP (fluorescence value 1269), the experimental group showed a 2.2-fold increase. This indicates that HSA-PEG... 16 -LNP has the strongest ability to transfect dendritic cells.

[0153] 2. Investigation of the targeting ability of nanoparticles to lymph nodes in animals

[0154] With PEG n -LNP (3.0%) HSA-PEG n -LNP (3.0%) The experimental group consisted of Moderna-LNP (a four-component nanoparticle, comparable to commercially available products) and the control group consisted of Moderna-LNP (a four-component nanoparticle, comparable to commercially available products) to investigate lymph node targeting.

[0155] Fluorescently labeled LNP nanoparticles were prepared by adding 300 μg of the near-infrared fluorescent dye DiR to a lipid-organic phase, a standard technique. Six- to eight-week-old female C57BL / 6 mice were subcutaneously injected with LNP (DiR dosage 50 ng / kg) at the base of the tail. Six hours later, the mice were sacrificed, and their left and right inguinal lymph nodes were removed, washed with physiological saline, and blotted dry with filter paper. Fluorescence intensity was detected using a small animal in vivo imaging system. Results are detailed below. Figure 6 Table 3.

[0156] Table 3. Specific fluorescence values ​​of nanoparticles enriched in lymph nodes.

[0157]

[0158] As shown in Table 3, the lymph node targeting ability of HSA-PEG is: 16 -LNP is significantly superior to PEG n -LNP and commercially available Moderna-LNP, and HSA-PEG 16 -LNP （3.0%） The lymph nodes have the strongest targeting ability.

[0159] 3. Antitumor effect of nanoparticles on melanoma

[0160] LNPs loaded with chicken ovalbumin OVA-mRNA were prepared according to the method in Example 2. The groups were arranged as follows: PBS solution as the negative control group, free OVA-mRNA as the positive control group 1, Moderna-LNP (four-component nanoparticles, comparable to commercially available products) as the positive control group 2, and PEG... 16 -LNP (3.0%) Positive control group 3, HSA-PEG 16 -LNP (3.0%) The experimental group consisted of mice with a mass ratio of 20:1 and 160 μL of albumin solution. Mice were subcutaneously inoculated with the B16-OVA cell line. On day 3 post-tumor implantation, the prepared nanoparticles or PBS solution were injected into the hind leg muscle of tumor-bearing C57BL / 6 mice at a dose of 20 μg mRNA. A second booster injection was administered on day 14. Tumor formation events were recorded daily, and the length and width of the tumor were measured every other day using calipers. The tumor volume was calculated using the formula: Tumor volume (cm²) 3= Tumor long diameter × Tumor short diameter 2 ×0.5. Note: OVA-mRNA is the messenger ribonucleic acid (mRNA) encoding ovalbumin (OVA). Ovalbumin is a large and complex glycoprotein that degrades into specific antigenic peptides within cells, eliciting a moderate immune response in the body. Therefore, it is widely used as a model antigen in immunological and biochemical research.

[0161] Results showed that HSA-PEG after treatment 16 -LNP （3%） Compared with the control group, tumor volume was significantly reduced. See details. Figure 7 Table 4.

[0162] Table 4. Tumor volume in mice 15 days after nanoparticle treatment (n = 7)

[0163]

[0164] It can be seen that HSA-PEG 16 -LNP has significant anti-tumor effects, HSA-PEG 16 -LNP （3%） Its antitumor activity is significantly superior to that of commercially available Moderna-LNP and PEG. 16 -LNP （3%） .

[0165] 4. Investigation into the activation of dendritic cells by nanoparticles

[0166] (1) Extraction and culture of dendritic cells (DCs)

[0167] Under aseptic conditions, the tibia and femur of C57BL / 6 mice were harvested, and other tissues were removed. The ends of the bones were cut off, and the bones were repeatedly rinsed with RPMI 1640 medium (without cytokines) using a syringe until the bone shaft turned white. The rinsing fluid was collected in centrifuge tubes and centrifuged at 1500 rpm for 5 min. The pellet was resuspended in RPMI 1640 medium containing the cytokines GM-CSF (20 ng / mL), IL-4 (10 ng / mL), fetal bovine serum (10%, v:v), penicillin (100 U / mL), and streptomycin (100 μg / mL), and then transferred to a cell culture flask. The cells were cultured at 37°C in a 5% CO2 incubator. The day of cell extraction was designated as day 0. Half the medium was replaced on days 3 and 5, and the suspension cells were collected on day 7, which were the primary dendritic cells (BMDCs).

[0168] (2) Evaluation of the rate of dendritic cell maturation

[0169] The expression of CD80 and CD86 was investigated by treating DCs with nanoparticles. Specifically, DCs cultured for 7 days were used, and 1×10⁻⁶ nanoparticles were applied to each well. 6 Cells were seeded in 6-well plates, and the following were added: PBS (negative control); mRNA expressing OVA protein (OVA-mRNA) as a separate mRNA control (similar to the free group, positive control 1); commercially available Moderna-LNP (positive control 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.

[0170] After incubation at 37°C for 24 hours, cells were collected by centrifugation, washed twice with PBS, and the expression of the DC surface activation markers CD80 and CD86 was detected by flow cytometry. Data are detailed in Table 5. It can be seen that the surface activation markers CD80 and CD86 of DCs did not change significantly in the PBS group, while the expression of HSA-PEG... 16 -LNP (3.0%) After treatment with DCs, the expression of both CD80 and CD86 was significantly increased (see Table 5 for details). The maturation rate was 2.7 times that of Moderna-LNP and PEG. 16 -LNP (3.0 %) It is 2.22 times that of PBS. OVA-mRNA, on the other hand, is similar to PBS.

[0171] The results showed that HSA-PEG 16 -LNP (3.0%) It can significantly stimulate the maturation of dendritic cells (DCs). The higher the activation rate of dendritic cell maturation, the stronger the activation effect on T cells, and the more beneficial it is for immunotherapy.

[0172] Table 5 Results of nanoparticle activation of dendritic cells

[0173]

[0174] This invention involves dissolving a lipid compound (functionalized low molecular weight PEG-maleimide derivative, Mal-PEGn-DMG) and commonly used nucleic acid delivery lipid composition materials (ionizable cationic lipids, phospholipid derivatives, cholesterol, PEGylated lipids, etc.) together in an organic solvent as the organic phase; dissolving nucleic acids (such as mRNA) in a buffer solution as the aqueous phase; and preparing the nucleic acid-loaded lipid nanoparticles mRNA-LNP by physical mixing or microfluidic methods using the above lipid organic phase and the nucleic acid-containing aqueous phase. Then, the nanoparticles are mixed with an albumin solution and incubated sequentially at room temperature, followed by overnight incubation at 4°C to allow the thiol groups on the albumin to couple with the maleimide on the nanoparticle surface. The nanoparticles are then purified by agarose gel column chromatography to obtain the albumin-coupled nucleic acid-loaded lipid nanoparticles HSA-mRNA-LNP. The steric hindrance introduced by the albumin coupling can reduce or overcome the immunogenicity of existing nanoparticles with an outer PEG coating.

Claims

1. An albumin-modified lipid nanoparticle or an albumin-modified drug-loaded lipid nanoparticle, characterized in that, The invention includes lipid nanoparticles and albumin on their surface; the raw materials for preparing the lipid nanoparticles include lipid compounds; the chemical structural formula of the lipid compounds is as follows: ； Wherein, R1 and R2 are -C(=O)OR, where R is a substituted or unsubstituted alkyl group; X is the residue part of the compound containing amino and carboxyl groups after substitution; Y includes ethylene glycol segments, hydrophilic functional polypeptide segments or PEG-like segments; Mal is a maleimide group; n is 1 to 5; in the ethylene glycol segments contained in Y, the number of repeating units of ethylene glycol is 1 to 25; in the raw materials for preparing lipid nanoparticles, the lipid compound accounts for 1.5% to 3% of the molar proportion of other raw materials.

2. The albumin-modified drug-loaded lipid nanoparticles according to claim 1, characterized in that, The medicine includes nucleic acids.

3. The method for preparing the albumin-modified lipid nanoparticles of claim 1 or the albumin-modified drug-loaded lipid nanoparticles, characterized in that, Lipid nanoparticles were prepared using lipid nanoparticle raw materials, and then albumin was modified on the lipid nanoparticles to obtain albumin-modified lipid nanoparticles. Drug-loaded lipid nanoparticles were prepared using lipid nanoparticle raw materials and drugs, and then albumin was modified on the drug-loaded lipid nanoparticles to obtain albumin-modified drug-loaded lipid nanoparticles.

4. The method for preparing albumin-modified lipid nanoparticles or albumin-modified drug-loaded lipid nanoparticles according to claim 3, characterized in that, The lipid nanoparticle raw material includes the lipid compound and other raw materials; the lipid compound accounts for 1.5% to 3% of the other raw materials in molar proportion; the lipid nanoparticles are mixed and incubated with albumin to obtain albumin-modified lipid nanoparticles; the drug-loaded lipid nanoparticles are mixed and incubated with albumin to obtain albumin-modified drug-loaded lipid nanoparticles.

5. The albumin-modified lipid nanoparticles of claim 1 or the albumin-modified drug-loaded lipid nanoparticles.

6. A biological product or drug, the active ingredient of which comprises the albumin-modified drug-loaded lipid nanoparticles of claim 1.

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