Disulfide bond-containing nucleic acid-lipid particle, medicine, application and preparation method

Abstract

A disulfide bond-containing nucleic acid-lipid particle includes: a nucleic acid; a cationic lipid, the molar percentage of which is in the range of 10%-70% with respect to all lipids in the disulfide bond-containing nucleic acid-lipid particle; cholesterol or a derivative thereof, the molar percentage of which is in the range of 5%-65% with respect to all lipids in the disulfide bond-containing nucleic acid-lipid particles; a phospholipid, wherein the molar percentage of the phospholipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle is in the range of 0%-50%; the molar percentage of the pegylated lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particles is in the range of 0%-20%; and a disulfide bond-containing lipid, wherein the mole percentage of the disulfide bond-containing lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle is in the range of 0.5%-60%. The invention also relates to a medicine comprising the nucleic acid-lipid particle containing the disulfide bond, an application of the nucleic acid-lipid particle containing the disulfide bond, and a preparation method of the nucleic acid-lipid particle containing the disulfide bond.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid drugs, and in particular to nucleic acid-lipid particles containing disulfide bonds, drugs comprising nucleic acid-lipid particles containing disulfide bonds, uses of nucleic acid-lipid particles containing disulfide bonds, and preparation methods of nucleic acid-lipid particles containing disulfide bonds. Background Art

[0002] Nucleic acid drugs generally include macromolecular nucleic acids formed by nucleotides and having a medical effect. Examples of nucleic acid drugs include, but are not limited to, Deoxyribonucleic Acid (DNA) drugs, Messenger Ribonucleic Acid (mRNA) drugs, Micro ribonucleic acid (miRNA) drugs, small interference ribonucleic acid (siRNA) drugs, antisense oligonucleotide (ASO) drugs, circular ribonucleic acid (circRNA) drugs, Self-amplifying RNA (saRNA) drugs, and Aptamer drugs, etc.

[0003] In recent years, the development of nucleic acid drugs has provided effective gene therapy means for the prevention and treatment of various diseases such as infectious diseases and tumors, protein replacement, protein inhibition, etc.

[0004] However, nucleic acids are extremely unstable in vitro and under physiological conditions and are easily degraded by nucleases in the air or blood. At the same time, nucleic acids carry negative charges and it is difficult to cross the cell membrane which also carries negative charges. The endosome-lysosome system also constitutes an important obstacle to the delivery of nucleic acid drugs. Therefore, one of the main difficulties faced by gene therapy is how to successfully deliver intact nucleic acids into the cytoplasm, and developing effective delivery vectors is one of the keys to releasing the therapeutic potential of nucleic acid drugs.

[0005] Lipid Nanoparticles (LNP) are an important class of nucleic acid delivery materials currently used clinically. During the preparation process of lipid nanoparticles, cationic lipids are protonated in acidic aqueous solutions and carry positive charges, and bind to negatively charged nucleic acids through electrostatic interactions to encapsulate the nucleic acids within the lipid nanoparticles. After encapsulation by lipid nanoparticles, the nucleic acids are protected extracellularly and are not affected by extracellular nucleases. Subsequently, through multiple steps such as endocytosis, lysosomal escape, and nucleic acid release, the nucleic acids can successfully enter the cytoplasm to achieve intracellular delivery and exert their functions.

[0006] In 2018, the first siRNA-LNP drug, Onpattro, was approved by the US Food and Drug Administration (FDA) for the treatment of transthyretin familial amyloid polyneuropathy. In 2020, two mRNA-LNP vaccines, BNT162b2 and mRNA-1273, received FDA emergency use authorization for the response to novel coronavirus infection. Thus, it can be seen that the LNP delivery platform plays a crucial role in nucleic acid drug delivery.

[0007] Although lipid nanoparticle delivery technology has been widely used in the field of nucleic acid drugs, lipid nanoparticles also face the barrier of the endosome-lysosome system, and the lysosomal sequestration of lipid nanoparticles remains a huge obstacle to delivery. When lipid nanoparticles enter cells through endocytosis and pinocytosis pathways, they need to escape from the endosome-lysosome system; otherwise, they will ultimately be degraded and destroyed by lysosomes.

[0008] Specifically, lipid nanoparticles entering cells through different pathways will exist in the cytoplasm in the form of various endosomes. As the endosome gradually matures, its internal environment changes from neutral to acidic. During this process, lipid nanoparticles will be protonated as the environmental pH decreases, and the charge changes from neutral to positive. The mature endosome will evolve into a lysosome, which has a lipid bilayer structure similar to the cell membrane, showing the asymmetry of the inner and outer lipid bilayers. The inner membrane of the lysosome usually contains a lot of negatively charged phosphatidylserine, making its inner membrane slightly negatively charged. The inner membrane of the lysosome and the protonated and positively charged lipid nanoparticles can fuse through the interaction of positive and negative charges, thereby releasing nucleic acids and achieving lysosomal escape.

[0009] The endosome-lysosome escape efficiency of nanoparticles is one of the limiting factors affecting the delivery efficiency of lipid nanoparticles. In fact, only 1-2% of the lipid nanoparticles phagocytosed by cells may successfully achieve lysosomal escape. According to research, only 1-2% of the siRNA in Onpattro may enter the cytoplasm.

[0010] Currently, the existing technical solutions for improving the cytoplasmic delivery efficiency of lipid nanoparticles mainly focus on improving the lysosomal escape efficiency of lipid nanoparticles. In addition, the structural geometric characteristics of lipid nanoparticles are also important factors affecting membrane fusion.

[0011] Based on the lysosomal escape mechanism and structural geometric characteristics of lipid nanoparticles, the existing technical solutions for improving the cytoplasmic delivery efficiency of lipid nanoparticles mainly include the following several types:

[0012] 1) By improving and optimizing cationic lipids, lipid nanoparticles with better membrane fusion ability and stronger lysosomal escape ability are developed; and

[0013] 2) Phytosterols are introduced to make the lipid nanoparticles present a polyhedral structure and enhance the membrane fusion ability.

[0014] However, excessive lysosomal rupture will release hydrolases, leading to autolysis and death of cells. Therefore, the lysosomal escape efficiency will not always increase. And:

[0015] (1) The process of developing new cationic lipids and carrying out large-scale production is very expensive and time-consuming; moreover, cationic lipids usually have toxic effects on cells. Therefore, when developing cationic lipids, it is necessary to balance the delivery efficiency and cytotoxicity; and

[0016] (2) The content of phytosterols is not rich enough, the supply is scarce and the production cost is high, which may limit its application in lipid nanoparticle formulations.

[0017] On the other hand, the research on dynamic covalent thiol-exchange chemistry has received attention in recent years and is considered an important technology to overcome the problem of "low lysosomal escape efficiency". However, existing lipid nanoparticles are taken up by cells through the classical endocytic pathway, and there is no publicly available information on the cell delivery of lipid nanoparticles through thiol-exchange mediation. If the delivery of lipid nanoparticles is to be changed to the thiol-exchange pathway, questions such as whether to introduce a thiol-exchange component that is completely different from the existing lipid components into the existing lipid nanoparticles, or to process the existing lipid components, how to introduce and process them, and whether the expected effects can be achieved after introduction and processing all remain to be answered.

[0018] Therefore, it is necessary to provide new technical solutions to solve one or more of the above problems, and / or other problems. Summary of the Invention

[0019] In view of this, an object of the present invention is to provide new technical solutions to solve one or more of the above problems, and / or other problems.

[0020] One aspect of an embodiment of the present application provides a nucleic acid-lipid particle containing a disulfide bond, which includes: a nucleic acid; a cationic lipid, the molar percentage of which in all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 10% to 70%; cholesterol or its derivative, the molar percentage of which in all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 5% to 65%; a phospholipid, the molar percentage of which in all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0% to 50%; a polyethylene glycolated lipid, the molar percentage of which in all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0% to 20%; and a lipid containing a disulfide bond, the molar percentage of which in all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0.5% to 60%.

[0021] In some embodiments, the lipid containing a disulfide bond includes a terminal disulfide bond.

[0022] In some embodiments, the lipid containing a disulfide bond includes one or more of a cationic lipid containing a disulfide bond, cholesterol or its derivative containing a disulfide bond, a phospholipid containing a disulfide bond, and a polyethylene glycolated lipid containing a disulfide bond.

[0023] In some embodiments, the lipid containing a disulfide bond includes a lipid modified with a disulfide functional group.

[0024] In some embodiments, the lipid containing a disulfide bond includes a lipid modified with a disulfide cyclic functional group.

[0025] In some embodiments, the lipid containing a disulfide bond includes a lipid modified with 1,2-dithiolane.

[0026] In some embodiments, the lipid containing a disulfide bond includes one or more of a cationic lipid modified with 1,2-dithiolane, cholesterol or its derivative modified with 1,2-dithiolane, a phospholipid modified with 1,2-dithiolane, and a polyethylene glycolated lipid modified with 1,2-dithiolane.

[0027] In some embodiments, the lipid containing a disulfide bond includes one or more of dioleoyl phosphatidylethanolamine modified with 1,2-dithiolane, cholesterol modified with 1,2-dithiolane, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 modified with 1,2-dithiolane.

[0028] In some embodiments, the lipid containing a disulfide bond includes a structural formula of

[0029] or one or more of the compounds.

[0030] In some embodiments, the molar percentage of the disulfide bond-containing lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particles ranges from 1% to 40%.

[0031] In some embodiments, the cationic lipid includes one or more of methyl (dilinoleoyl) 4-(N,N-dimethylamino)butyrate, trimethyl-2,3-dioleyloxypropylammonium bromide, heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, and ((4-hydroxybutyl)azaalkanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0032] In some embodiments, the cholesterol or its derivative includes cholesterol.

[0033] In some embodiments, the phospholipid includes distearoyl phosphatidylcholine and / or dioleoyl phosphatidylethanolamine.

[0034] In some embodiments, the polyethylene glycolated lipid includes 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide and / or 1,2-dimyristoyl-RAC-glycerol-3-methoxypolyethylene glycol 2000.

[0035] In some embodiments, the molar percentage of the cationic lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particles ranges from 25% to 60%.

[0036] In some embodiments, the molar percentage of the cholesterol or its derivative relative to all lipids in the disulfide bond-containing nucleic acid-lipid particles ranges from 20% to 45%.

[0037] In some embodiments, the molar percentage of the phospholipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particles ranges from 0% to 15%.

[0038] In some embodiments, the molar percentage of the polyethylene glycolated lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particles ranges from 0% to 2%.

[0039] In some embodiments, the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particles ranges from 10:1 to 100:1.

[0040] In some embodiments, the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particles ranges from 20:1 to 40:1.

[0041] In some embodiments, the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particles is 40:1.

[0042] In some embodiments, the nucleic acid includes one or more of deoxyribonucleic acid, messenger ribonucleic acid, micro ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleotide, circular ribonucleic acid, self-amplifying ribonucleic acid, and nucleic acid aptamer.

[0043] In some embodiments, the nucleic acid includes one or more of green fluorescent protein messenger ribonucleic acid, firefly luciferase messenger ribonucleic acid, and severe acute respiratory syndrome coronavirus 2 spike protein messenger ribonucleic acid.

[0044] In some embodiments, the particle size of the disulfide bond-containing nucleic acid-lipid particle ranges from 50 nanometers to 300 nanometers.

[0045] In some embodiments, the particle size of the disulfide bond-containing nucleic acid-lipid particle ranges from 110 nanometers to 210 nanometers.

[0046] Another aspect of the embodiments of the present application relates to a drug, which comprises a pharmaceutical carrier and the disulfide bond-containing nucleic acid-lipid particle as described in the present application.

[0047] In some embodiments, the drug is one or more of an infectious disease vaccine, a cancer vaccine, a tumor immunotherapy drug, a messenger ribonucleic acid drug based on direct encoding of a protein, a protein replacement drug, and a protein inhibition drug.

[0048] In some embodiments, the drug is one or more of a deoxyribonucleic acid drug, a messenger ribonucleic acid drug, a micro ribonucleic acid drug, a small interfering ribonucleic acid drug, an antisense oligonucleotide drug, a circular ribonucleic acid drug, a self-amplifying ribonucleic acid drug, and a nucleic acid aptamer drug.

[0049] In some embodiments, the drug is a severe acute respiratory syndrome coronavirus 2 vaccine.

[0050] In some embodiments, the drug is a humoral immune drug.

[0051] In some embodiments, the drug is a messenger ribonucleic acid vaccine.

[0052] Another aspect of the embodiments of the present application relates to the use of the disulfide bond-containing nucleic acid-lipid particle as described in the present application in the preparation of a product for introducing nucleic acid into cells.

[0053] In some embodiments, the cell is a mammalian cell.

[0054] Another aspect of the embodiments of the present application relates to the use of the disulfide bond-containing nucleic acid-lipid particle as described in the present application in the preparation of a product for in vivo delivery of nucleic acid to be administered to a mammal.

[0055] Another aspect of the embodiments of the present application relates to the use of the nucleic acid-lipid particles containing disulfide bonds described in the present application in the preparation of a medicament for administration to a mammal.

[0056] In some embodiments, the medicament is one or more of an infectious disease vaccine, a cancer vaccine, a tumor immunotherapy drug, a messenger ribonucleic acid drug based on directly encoding a protein, a protein replacement drug, and a protein inhibition drug.

[0057] In some embodiments, the medicament is one or more of a deoxyribonucleic acid drug, a messenger ribonucleic acid drug, a micro ribonucleic acid drug, a small interfering ribonucleic acid drug, an antisense oligonucleotide drug, a circular ribonucleic acid drug, a self-amplifying ribonucleic acid drug, and a nucleic acid aptamer drug.

[0058] In some embodiments, the medicament is a novel coronavirus vaccine.

[0059] In some embodiments, the medicament is a humoral immune drug.

[0060] In some embodiments, the medicament is a messenger ribonucleic acid vaccine.

[0061] Another aspect of the embodiments of the present application relates to a method for preparing the nucleic acid-lipid particles containing disulfide bonds described in the present application, which includes:

[0062] Dissolving the cationic lipid, the cholesterol or its derivative, the phospholipid, the polyethylene glycolated lipid, and the lipid containing disulfide bonds in an organic solvent to obtain an organic phase lipid solution;

[0063] Dissolving the nucleic acid in an acidic buffer solution to obtain an aqueous phase nucleic acid solution; and

[0064] Mixing the organic phase lipid solution with the aqueous phase nucleic acid drug solution to obtain the nucleic acid-lipid particles containing disulfide bonds.

[0065] In some embodiments, the organic phase lipid solution and the aqueous phase nucleic acid drug solution are microfluidically mixed, and the total flow rate ranges from 1 mL / min to 30 mL / min.

[0066] In some embodiments, the organic phase lipid solution and the aqueous phase nucleic acid drug solution are mixed with a pipette.

[0067] In some embodiments, the preparation method includes purifying after mixing the organic phase lipid solution with the aqueous phase nucleic acid drug solution to obtain the nucleic acid-lipid particles containing disulfide bonds.

[0068] In some embodiments, the preparation method includes modifying a disulfide functional group on a raw material lipid to obtain the lipid containing disulfide bonds.

[0069] Another aspect of the embodiments of the present application relates to a method for preventing and treating diseases, which includes administering the drug described in the present invention to a mammal.

[0070] Another aspect of the embodiments of the present application relates to a method for antiviral and / or cell infection prevention, which includes administering the drug described in the present invention to a mammal.

[0071] Another aspect of the embodiments of the present application relates to a method for introducing nucleic acid into cells, which includes: contacting the cells with the disulfide bond-containing nucleic acid-lipid particles described in the present application.

[0072] Another aspect of the embodiments of the present application relates to a method for introducing nucleic acid into cells, which includes: contacting the cells with the disulfide bond-containing nucleic acid-lipid particles described in the present application in vitro.

[0073] Another aspect of the embodiments of the present application relates to a method for in vivo delivery of nucleic acid, which includes administering the disulfide bond-containing nucleic acid-lipid particles described in the present application to a mammal.

[0074] Another aspect of the embodiments of the present application relates to a method for using the disulfide bond-containing nucleic acid-lipid particles described in the present application for in vivo delivery of nucleic acid, which includes administering the disulfide bond-containing nucleic acid-lipid particles to a mammal.

[0075] Another aspect of the embodiments of the present application relates to a method for treating a disease or disorder in a mammal, which includes administering an effective therapeutic dose of the disulfide bond-containing nucleic acid-lipid particles described in the present application to the mammal.

[0076] Another aspect of the embodiments of the present application relates to a method for using the disulfide bond-containing nucleic acid-lipid particles described in the present application for treating a disease or disorder in a mammal, which includes administering an effective therapeutic dose of the disulfide bond-containing nucleic acid-lipid particles to the mammal.

[0077] In some embodiments, the disease or disorder is selected from viral infection, tumor.

[0078] Another aspect of the embodiments of the present application relates to the use of the disulfide bond-containing nucleic acid-lipid particles described in the present application in gene therapy.

[0079] In some embodiments, the gene therapy includes one or more of deoxyribonucleic acid therapy, messenger ribonucleic acid therapy, micro ribonucleic acid therapy, small interfering ribonucleic acid therapy, antisense oligonucleotide therapy, circular ribonucleic acid therapy, self-amplifying ribonucleic acid therapy, and nucleic acid aptamer therapy.

[0080] The technical features of the embodiments in this application can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in each embodiment are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0081] In order to make the purpose, technical solutions and advantages of this application more clear and understandable, the following further details this application in combination with the accompanying drawings and specific embodiments, but this does not limit the present invention within the scope of the described embodiments. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Brief Description of the Drawings

[0082] Figure 1 Shows the transmission electron microscope (TEM) images and cryo-electron microscopy (Cryo-EM) images of SLNP-A9 in Examples 1-2.

[0083] Figure 2 Shows the microscope images and flow analysis statistical charts of each nucleic acid-lipid particle with different molar percentages of disulfide-containing lipids and different doses of green fluorescent protein (GFP) messenger ribonucleic acid (mRNA) on human embryonic kidney cells (293T cells) in Example 2-1.

[0084] Figure 3 Shows the fluorescence intensity of green fluorescent protein of each nucleic acid-lipid particle based on the commercial lipid formulations of mRNA-1273 and BNT162b2 on 293T cells in Example 2-1.

[0085] Figure 4 Shows the confocal images and flow analysis diagrams of the uptake of each nucleic acid-lipid particle and naked Cy5-GFP mRNA by each cell in Example 2-2.

[0086] Figure 5 Shows the flow analysis diagram of the mechanism of each cell taking up each disulfide-containing nucleic acid-lipid particle in Example 2-2.

[0087] Figure 6 Shows the cytotoxicity test results of each disulfide-containing lipid and each nucleic acid-lipid particle in Example 2-3.

[0088] Figure 7 The enzyme-linked immunosorbent assay (ELISA) results of the expression levels of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein in 293T cells by each nucleic acid-lipid particle in Examples 2-4 are shown.

[0089] Figure 8 The transfection effect diagrams of Firefly Luciferase Messenger Ribonucleic Acid (FLuc mRNA) of each nucleic acid-lipid particle in Balb / c mice in Example 3 are shown.

[0090] Figure 9 The transfection effect diagrams of Firefly Luciferase Messenger Ribonucleic Acid (FLuc mRNA) of each nucleic acid-lipid particle administered by various administration routes in mice in Example 3 are shown.

[0091] Figure 10 The antibody levels produced in mice after the first immunization and the second immunization by the SARS-CoV-2 messenger ribonucleic acid vaccine based on each nucleic acid-lipid particle in Example 4 are shown.

[0092] Figure 11 The analysis results of the serum biochemical indexes of each mouse after injection of each vaccine in Example 5 are shown. Detailed implementation manners

[0093] One aspect of the embodiments of the present application provides a nucleic acid-lipid particle containing a disulfide bond, which includes: a nucleic acid; a cationic lipid, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 10% to 70%; cholesterol or its derivative, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 5% to 65%; a phospholipid, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0% to 50%; a polyethylene glycolated lipid, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0% to 20%; and a lipid containing a disulfide bond, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0.5% to 60%.

[0094] Unless otherwise specifically stated, the numerical values in this application may include errors such as measurement errors, precision errors, and measurement errors, for example, errors within ±5%. For example, 10% may include numerical values within the range of 10%×(1±5%), that is, numerical values within the interval of 9.5% to 10.5%, such as 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10.0%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, and so on.

[0095] In this application, unless otherwise specifically stated, the numerical range may include any sub-range therein. For example, 0.5%-60% may include 0.5%-5%, 0.5%-10%, 0.5%-15%, 0.5%-20%, 0.5%-25%, 3%-8%, 5%-30%, 15%-40%, 25%-50%, 35%-60%, 2%-38%, 1%-40%, 50%-60%, and so on.

[0096] The disulfide bond-containing nucleic acid-lipid particles have low cytotoxicity and high biosafety. The disulfide bond-containing nucleic acid-lipid particles can help the nucleic acid to be efficiently taken up by cells and have good transfection effects at the cellular level and in vivo.

[0097] The disulfide bond-containing nucleic acid-lipid particles have high delivery efficiency, high nucleic acid expression efficiency, good biosafety, can produce high antibody levels, and have advantages such as reducing the dosage and / or increasing the protein expression level in biomedical applications.

[0098] In some embodiments, the disulfide bond-containing nucleic acid-lipid particles can be efficiently taken up by cells through a thiol-mediated cellular uptake mechanism. Through a dynamic thiol exchange reaction, the disulfide bond of the disulfide bond-containing nucleic acid-lipid particles can covalently bind to the thiol groups on the outer surface of the cell membrane and be rapidly transported into the cytoplasm. The disulfide bond-containing lipid can also be called a thiol lipid. The disulfide bond-containing nucleic acid-lipid particles can also be called thiol lipid nanoparticles.

[0099] Compared with traditional lipid nanoparticles that deliver nucleic acids through an endocytosis delivery method, the disulfide bond-containing nucleic acid-lipid particles directly deliver nucleic acids into the cytoplasm, which can effectively avoid the capture and degradation by endosomes and lysosomes, solve the problem of low lysosomal escape efficiency of existing lipid nanoparticles, and greatly improve the nucleic acid delivery efficiency.

[0100] Moreover, the disulfide bond-containing nucleic acid-lipid particles can help prevent excessive lysosomal rupture and release of hydrolases and prevent cell autolysis and death.

[0101] The disulfide bond-containing nucleic acid-lipid particles do not require the development of new cationic lipids, are suitable for large-scale production processes, have relatively low costs, and take less time.

[0102] The raw materials of the disulfide bond-containing nucleic acid-lipid particles are abundant, the supply is secure, and the production cost is relatively low.

[0103] Referring to the experimental examples described later, compared with traditional lipid nanoparticles, the cell uptake ability, protein expression ability, etc. of the disulfide bond-containing nucleic acid-lipid particles in the examples of the present application have been significantly improved. Therefore, the technical solution of the present application can effectively solve the problem of low lysosomal escape efficiency of traditional lipid nanoparticles.

[0104] In some embodiments, a disulfide bond-containing lipid is added to the components of existing lipid nanoparticles to obtain disulfide bond-containing nucleic acid-lipid particles. After adding the disulfide bond-containing lipid to the basic lipid nanoparticle system, the original basic lipid nanoparticle delivery system via the endocytosis pathway can be transformed into the disulfide bond-containing nucleic acid-lipid particle delivery system via the thiol exchange pathway. The disulfide bond-containing nucleic acid-lipid particles directly deliver nucleic acids into the cytoplasm through the thiol exchange pathway, which can effectively solve problems such as lysosomal sequestration of traditional lipid nanoparticles.

[0105] In the present application, a disulfide bond can refer to a functional group with the structure R-S-S-R', where R and R' can be the same or different. Examples of R and R' can independently include a hydrocarbon group (such as methylene -CH-, methylene -CH 2 -), a sulfide group -S-, an aromatic group (such as a phenyl group), a chemical bond, and so on. The disulfide bond can be a part of a cyclic functional group, a linear functional group, and / or a chain-like functional group. Examples of the cyclic functional group containing a disulfide bond include, but are not limited to, 1,2-dithiolane, where R and R' are respectively methylene -CH-, methylene -CH 2 -. When R and R' are sulfide groups -S-, chemical bonds, the disulfide bond can be, for example, -S-S-S-.

[0106] The disulfide bond-containing lipid can have one disulfide bond or multiple disulfide bonds. For example, it can include a poly-1,2-dithiolane structural unit formed by polymerization of multiple 1,2-dithiolanes.

[0107] In some embodiments, the disulfide bond-containing lipid includes a terminal disulfide bond. The terminal disulfide bond in the disulfide bond-containing lipid can covalently bind to the sulfhydryl group on the outer surface of the cell membrane, which helps the disulfide bond-containing nucleic acid-lipid particles to be rapidly transported into the cytoplasm and be efficiently taken up by the cells.

[0108] The disulfide bond-containing lipid may have one or more ends. Correspondingly, the disulfide bond-containing lipid may include one or more disulfide bonds located at one or more ends.

[0109] The disulfide bond-containing cyclic functional group may be located at the end of the disulfide bond-containing lipid. The disulfide bond-containing cyclic functional group located at the end of the disulfide bond-containing lipid may be connected to other parts of the disulfide bond-containing lipid at only one place.

[0110] The disulfide bond-containing lipid may be prepared from a raw material lipid. The raw material lipid may be an existing lipid, which includes, but is not limited to, existing cationic lipids, existing cholesterol or its derivatives, existing phospholipids, and existing polyethylene glycolated lipids, which may help avoid the development of new cationic lipids, cholesterol or its derivatives, phospholipids, and polyethylene glycolated lipids.

[0111] The raw material lipid may include a raw material cationic lipid, a raw material cholesterol or its derivative, a raw material phospholipid, and / or a raw material polyethylene glycolated lipid. Lipids derived from the raw material cationic lipid, the raw material cholesterol or its derivative, the raw material phospholipid, and the raw material polyethylene glycolated lipid and containing disulfide bonds may be respectively referred to as a disulfide bond-containing cationic lipid, a disulfide bond-containing cholesterol or its derivative, a disulfide bond-containing phospholipid, and a disulfide bond-containing polyethylene glycolated lipid.

[0112] In some embodiments, the disulfide bond-containing lipid includes one or more of a disulfide bond-containing cationic lipid, a disulfide bond-containing cholesterol or its derivative, a disulfide bond-containing phospholipid, and a disulfide bond-containing polyethylene glycolated lipid.

[0113] The raw material cationic lipid, the raw material cholesterol or its derivative, the raw material phospholipid, and the raw material polyethylene glycolated lipid may be the same as or different from the cationic lipid, the cholesterol or its derivative, the phospholipid, and the polyethylene glycolated lipid in the disulfide bond-containing nucleic acid-lipid particle, respectively.

[0114] The disulfide bond-containing lipid may be obtained by modifying the raw material lipid with a disulfide compound. The disulfide bond-containing lipid obtained by modifying the raw material lipid with a disulfide compound may be referred to as a lipid with a modified disulfide functional group.

[0115] In some embodiments, the disulfide bond-containing lipid includes a lipid with a modified disulfide functional group.

[0116] The preparation process of the lipid with a modified disulfide functional group is relatively simple, the cost is relatively low, and it is suitable for large-scale production.

[0117] The dithiol compound may include a dithiol cyclic functional group. The lipid containing a disulfide bond obtained by modifying a raw material lipid with the dithiol cyclic functional group may be referred to as a lipid modified with a dithiol cyclic functional group.

[0118] In some embodiments, the lipid containing a disulfide bond includes a lipid modified with a dithiol cyclic functional group.

[0119] The lipid modified with a dithiol cyclic functional group may include a cyclic functional group containing a disulfide bond. The cyclic functional group containing a disulfide bond may be a three-membered ring, a four-membered ring, a five-membered ring, a six-membered ring, and so on. The cyclic functional group containing a disulfide bond may be a dithiol heterocycle containing two sulfur atoms.

[0120] Examples of the dithiol cyclic functional group may include, but are not limited to, 1,2-dithiolane.

[0121] In some embodiments, the lipid containing a disulfide bond includes a lipid modified with 1,2-dithiolane.

[0122] The raw materials of the lipid modified with 1,2-dithiolane are abundant, the raw material cost is relatively low, the preparation process is relatively simple, and the preparation cost is relatively low, which is suitable for large-scale production.

[0123] The lipid containing a disulfide bond can be prepared by conventional methods in the art. For example, modifying 1,2-dithiolane on a raw material lipid can be achieved through a condensation reaction between the carboxyl group of lipoic acid and the amino group / hydroxyl group of the raw material lipid. This condensation reaction is a classical organic reaction and only requires one-step reaction, so laboratories and factories with a synthetic basis can complete this reaction.

[0124] For example, mixing lipoic acid containing 1,2-dithiolane with phosphatidyl ethanolamine DOPE for an amide condensation reaction can obtain 1,2-dithiolane-modified dioleoyl phosphatidylethanolamine (S-DOPE).

[0125] The lipid containing a disulfide bond can directly prepare the nucleic acid-lipid particles without subsequent coupling or insertion steps, etc. The preparation process is simple and suitable for large-scale preparation and production.

[0126] The lipid containing a disulfide bond has universality and can be used to prepare nucleic acid-lipid particles containing disulfide bonds with various different formulations and different ratios. Therefore, the technical solution of the present application can enrich the development strategies of lipid nanoparticles and / or shorten the R & D time.

[0127] In some embodiments, the disulfide bond-containing lipid includes one or more of a cationic lipid modified with 1,2-dithiolane, cholesterol or a derivative thereof modified with 1,2-dithiolane, a phospholipid modified with 1,2-dithiolane, and a polyethylene glycolated lipid modified with 1,2-dithiolane.

[0128] In some embodiments, the disulfide bond-containing lipid includes one or more of 1,2-dithiolane-modified dioleoylphosphatidylethanolamine (S-DOPE), 1,2-dithiolane-modified cholesterol (S-CHOL), and 1,2-dithiolane-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 (S-DSPE-PEG2K).

[0129] In some embodiments, the disulfide bond-containing lipid includes any one of 1,2-dithiolane-modified dioleoylphosphatidylethanolamine (S-DOPE), 1,2-dithiolane-modified cholesterol (S-CHOL), and 1,2-dithiolane-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 (S-DSPE-PEG2K).

[0130] In some embodiments, the disulfide bond-containing lipid includes any two of 1,2-dithiolane-modified dioleoylphosphatidylethanolamine (S-DOPE), 1,2-dithiolane-modified cholesterol (S-CHOL), and 1,2-dithiolane-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 (S-DSPE-PEG2K), such as 1,2-dithiolane-modified dioleoylphosphatidylethanolamine (S-DOPE) and 1,2-dithiolane-modified cholesterol (S-CHOL).

[0131] In some embodiments, the disulfide bond-containing lipid includes 1,2-dithiolane-modified dioleoylphosphatidylethanolamine (S-DOPE), 1,2-dithiolane-modified cholesterol (S-CHOL), and 1,2-dithiolane-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 (S-DSPE-PEG2K).

[0132] The structural formula of 1,2-dithiolane-modified dioleoylphosphatidylethanolamine may be

[0133] The structural formula of 1,2-dithiolane-modified cholesterol may be

[0134] The structural formula of 1,2-dithiolane-modified distearoylphosphatidylethanolamine-polyethylene glycol 2000 may be

[0135] In some embodiments, the disulfide bond-containing lipid includes a structural formula of or one or more of the compounds of .

[0136] In some embodiments, the molar percentage of the disulfide bond-containing lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle ranges from 1% to 40%.

[0137] The molar percentage of the disulfide bond-containing lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be the percentage of the number of moles of the disulfide bond-containing lipid in the sum of the number of moles of all lipids in the disulfide bond-containing nucleic acid-lipid particle.

[0138] The molar percentage of the disulfide bond-containing lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may range, for example, from 2% to 40%, from 25% to 40%, from 20% to 40%, from 15% to 30%, from 30% to 40%, from 35% to 40%, from 1% to 36.3%, or from 10% to 20%, etc.

[0139] The molar percentage of the disulfide bond-containing lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be, for example, 1%, 10%, 20%, 30%, 4.8%, 9.1%, 13%, 16.6%, 23%, 21.4%, 22.3%, 36.3%, 1.5%, 2.5%, 3.5%, etc.

[0140] The disulfide bond-containing nucleic acid-lipid particle may comprise one or more of the cationic lipids. In the present application, the term "cationic lipid" may refer to a lipid that carries a net positive charge at a selected pH value, such as physiological pH (e.g., pH of about 7.0). Descriptions of cationic lipids can be found in Chinese Patent Publication No. CN102119217B; US Patent Application Publication Nos. 20060083780 and 20060240554; US Patents Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992; and International Patent Application Publication No. WO 96 / 10390; etc.

[0141] In some embodiments, the cationic lipid and the raw material cationic lipid may each independently include one or more of methyl (dilinoleoyl) 4-(N,N-dimethylamino) butyrate (Dlin-MC3-DMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), 9-heptadecanyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino) octanoate (SM-102), ((4-hydroxybutyl)aza-dialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315).

[0142] The cholesterol or its derivative, and the raw material cholesterol or its derivative may each be a neutral lipid. Examples of the cholesterol derivative and the raw material cholesterol derivative may each independently include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and any mixture thereof. The synthesis of cholesteryl-2'-hydroxyethyl ether may be referred to Chinese Patent Publication No. CN102119217B, etc.

[0143] In some embodiments, the cholesterol or its derivative, and the raw material cholesterol or its derivative each include cholesterol.

[0144] In the present application, unless otherwise specifically stated, the term "neutral lipid" may refer to a lipid that exists in an uncharged or neutral zwitterionic form at a selected pH value.

[0145] The phospholipid and the raw material phospholipid may be neutral lipids or amphiphilic lipids. The phospholipid and the raw material phospholipid may each independently include, but are not limited to, dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), palmitoyl oleoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyl oleoyl-phosphatidylethanolamine (SOPE), egg yolk phosphatidylcholine (EPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, or any mixture of the foregoing substances.

[0146] As amphiphilic lipids, the hydrophobic moieties of the phospholipids and the starting phospholipids can be oriented towards the hydrophobic phase, while the hydrophilic moieties can be oriented towards the aqueous phase. The hydrophilic nature of the phospholipids and the starting phospholipids can be derived from polar or charged groups such as sugars, phosphate groups, carboxyl groups, sulfate groups, amino groups, mercapto groups, nitro groups, hydroxyl groups, and other similar groups. The hydrophobicity of the phospholipids and the starting phospholipids can be conferred by including non-polar groups, which include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and long-chain saturated and unsaturated aliphatic hydrocarbon groups substituted by one or more aromatic, alicyclic, or heterocyclic groups.

[0147] In some embodiments, the phospholipids and the starting phospholipids can respectively include dipalmitoyl phosphatidylcholine (DPPC), and / or dioleoyl phosphatidylethanolamine (DOPE).

[0148] The PEGylated lipids and the starting PEGylated lipids can respectively be lipids including a poly(ethylene glycol) (PEG) moiety. The PEG and the starting PEGylated lipids can respectively be linear, water-soluble polymers of ethylene glycol repeating units having two terminal hydroxyl groups. The PEG moiety can include an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In certain embodiments, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). The PEG moiety can be classified by its molecular weight. For example, PEG2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. The PEGylated lipids and the starting PEGylated lipids can be beneficial for inhibiting the aggregation of the disulfide bond-containing nucleic acid-lipid particles.

[0149] In some embodiments, the PEGylated lipids and the starting PEGylated lipids can respectively include 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), and / or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2K).

[0150] In some embodiments, the molar percentage of the cationic lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particles ranges from 25% to 60%.

[0151] The molar percentage of the cationic lipid relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle can be the percentage of the number of moles of the cationic lipid in the sum of the number of moles of all the lipids in the disulfide bond-containing nucleic acid-lipid particle.

[0152] The molar percentage of the cationic lipid relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle can be in a range such as 35%-60%, 25%-50%, 25%-40%, 25%-30%, 30%-60%, 35%-50%, 27.3%-59%, or 30%-50%, etc.

[0153] The molar percentage of the cationic lipid relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle can be, for example, 50%, 59%, 45%, 40%, 47.6%, 45.4%, 43.5%, 41.7%, 38.5%, 42.9%, 33.3%, 30%, 27.3%, 49.5%, 49%, 46.3%, 38.7%, and so on.

[0154] In some embodiments, the molar percentage of the cholesterol or its derivative relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle is in the range of 20%-45%.

[0155] The molar percentage of the cholesterol or its derivative relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle can be the percentage of the number of moles of the cholesterol or its derivative in the sum of the number of moles of all the lipids in the disulfide bond-containing nucleic acid-lipid particle.

[0156] The molar percentage of the cholesterol or its derivative relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle can be in a range such as 21%-44%, 25%-40%, 30%-44%, 35%-40%, 21.4%-42.9%, or 40%-44%, etc.

[0157] The molar percentage of the cholesterol or its derivative relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle can be, for example, 32.1%, 38.5%, 35.6%, 42.7%, 37.7%, 38.1%, 27.3%, 30%, 33.3%, 21.4%, 42.9%, 29.6%, 30.8%, 33.5%, 35%, 36.7%, 28.5%, 23.5%, and so on.

[0158] In some embodiments, the molar percentage of the phospholipid relative to all the lipids in the disulfide bond-containing nucleic acid-lipid particle is in the range of 0%-15%.

[0159] The molar percentage of the phospholipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be the percentage of the number of moles of the phospholipid in the sum of the number of moles of all lipids in the disulfide bond-containing nucleic acid-lipid particle.

[0160] The molar percentage of the phospholipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be in a range such as 0%-15%, 0%-1%, 0%-2%, 0%-3%, 0%-4%, 0%-5%, 0%-6%, 0%-7%, 0%-8%, 10%-15%, 10%-14%, 10%-13%, 10%-12%, 10%-11%, 5%-10%, 6%-11%, 8%-12%, 9%-11%, 0%-14.3%, or 9%-10%, etc.

[0161] The molar percentage of the phospholipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be, for example, 10%, 0%, 9.5%, 9.1%, 8.7%, 8.3%, 8%, 7.7%, 14.2%, 14.3%, 11.1%, 9.1%, 9.9%, 9.8%, 9.4%, 7.8%, and so on.

[0162] In some embodiments, the molar percentage of the polyethylene glycolylated lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle is in the range of 0%-2%.

[0163] The molar percentage of the polyethylene glycolylated lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be the percentage of the number of moles of the polyethylene glycolylated lipid in the sum of the number of moles of all lipids in the disulfide bond-containing nucleic acid-lipid particle.

[0164] The molar percentage of the polyethylene glycolylated lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be in a range such as 0%-2%, 0%-1%, 0%-1.5%, 1%-2%, 0%-1.6%, or 1.5%-2%.

[0165] The molar percentage of the polyethylene glycolylated lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle may be, for example, 1.3%, 1.5%, 1.6%, 0%, 1.2%, 1.4%, and so on.

[0166] In some embodiments, the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle is in the range of 10:1 to 100:1. Thus, it can be beneficial for effective encapsulation of the nucleic acid, and can help prevent waste of lipids and / or generation of biological toxicity.

[0167] The mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle can be the ratio of the sum of the masses of all lipids in the disulfide bond-containing nucleic acid-lipid particle to the mass of the nucleic acid.

[0168] The mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle can be in the range of, for example, 10:1 to 100:1, 15:1 to 95:1, 25:1 to 85:1, 35:1 to 75:1, 45:1 to 65:1, 55:1 to 60:1, 20:1 to 95:1, 20:1 to 40:1, 30:1 to 70:1, or 60:1 to 80:1, etc.

[0169] In some embodiments, the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle is in the range of 20:1 to 40:1.

[0170] The mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle can be, for example, 10:1, 100:1, 15:1, 95:1, 25:1, 85:1, 35:1, 75:1, 45:1, 65:1, 55:1, 60:1, 20:1, 30:1, 40:1, 70:1, or 80:1, etc.

[0171] In some embodiments, the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle is about 40:1.

[0172] The nucleic acid can contain a polymer of at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form. The nucleic acid can include oligonucleotides and / or polynucleotides, can have a fragment containing up to 60 nucleotides, which is commonly referred to as an oligonucleotide, and / or a longer fragment, which is called a polynucleotide. The nucleic acid can include analogs, such as phosphorothioates, aminophosphates, methylphosphates, etc.

[0173] The nucleic acid can have different length ranges. For example, the length range of the nucleic acid can be about 1,000 - about 100,000 nucleotides, about 10 - about 100 nucleotides, about 10 - about 60 nucleotides, about 15 - about 60 nucleotides, about 20 - about 50 nucleotides, about 15 - about 30 nucleotides, or about 20 - about 30 nucleotides.

[0174] In some embodiments, the nucleic acid includes one or more of deoxyribonucleic acid, messenger ribonucleic acid, micro ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleotides, circular ribonucleic acid, self-amplifying ribonucleic acid, and nucleic acid aptamers.

[0175] In some embodiments, the nucleic acid includes any one of deoxyribonucleic acid, messenger ribonucleic acid, micro ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleotide, circular ribonucleic acid, self-amplifying ribonucleic acid, and nucleic acid aptamer.

[0176] In some embodiments, the nucleic acid includes two or more of deoxyribonucleic acid, messenger ribonucleic acid, micro ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleotide, circular ribonucleic acid, self-amplifying ribonucleic acid, and nucleic acid aptamer. For example, the disulfide bond-containing nucleic acid-lipid particles can simultaneously load messenger ribonucleic acid and small interfering ribonucleic acid.

[0177] In some embodiments, the nucleic acid includes one or more of Green Fluorescent Protein Messenger Ribonucleic Acid (GFP mRNA), Firefly Luciferase Messenger Ribonucleic Acid (FLuc mRNA), and SARS-CoV-2 S Protein Messenger Ribonucleic Acid (SP mRNA).

[0178] The Green Fluorescent Protein Messenger Ribonucleic Acid (GFP mRNA) and the Firefly Luciferase Messenger Ribonucleic Acid (FLuc mRNA) can be referred to as reporter genes.

[0179] Referring to the experimental examples described later, in vitro and in vivo studies using reporter genes such as GFP mRNA and FLuc mRNA can show that after thiol-mediated delivery, the disulfide bond-containing nucleic acid-lipid particle delivery system produces a stronger optical signal than the conventional lipid nanoparticle delivery system, indicating that the disulfide bond-containing nucleic acid-lipid particles can deliver more nucleic acids to the cytoplasm to play a role.

[0180] In some embodiments, the sequence information of the Green Fluorescent Protein Messenger Ribonucleic Acid (GFP mRNA), the Firefly Luciferase Messenger Ribonucleic Acid (FLuc mRNA), and the SARS-CoV-2 S Protein Messenger Ribonucleic Acid (SP mRNA) can be as shown in the following table respectively.

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187] The nucleic acid-lipid particles containing disulfide bonds can be nanoparticles. The nucleic acid-lipid particles containing disulfide bonds can be spherical. The particle size of the nucleic acid-lipid particles containing disulfide bonds can be detected using Dynamic Light Scattering (DLS), Transmission Electron Microscope (TEM), and / or Cryo-Electron Microscopy (Cryo-EM). The particle size of the nucleic acid-lipid particles containing disulfide bonds can be measured in nanometers. The particle size of the nucleic acid-lipid particles containing disulfide bonds can be the average particle size. The particle size of the nucleic acid-lipid particles containing disulfide bonds can be the average diameter.

[0188] In some embodiments, the particle size of the nucleic acid-lipid particles containing disulfide bonds is in the range of 50 nanometers to 300 nanometers.

[0189] The particle size of the nucleic acid-lipid particles containing disulfide bonds can be in ranges such as 50 nanometers to 300 nanometers, 50 nanometers to 200 nanometers, 50 nanometers to 100 nanometers, 60 nanometers to 250 nanometers, 70 nanometers to 280 nanometers, 100 nanometers to 250 nanometers, 113 nanometers to 204 nanometers, 113.0 nanometers to 204.4 nanometers, or 110 nanometers to 210 nanometers, etc.

[0190] In some embodiments, the particle size of the disulfide bond-containing nucleic acid-lipid particles ranges from 110 nanometers to 210 nanometers.

[0191] The particle size of the disulfide bond-containing nucleic acid-lipid particles can be, for example, 117.5 nanometers, 115.1 nanometers, 120.5 nanometers, 123.7 nanometers, 117.3 nanometers, 121.1 nanometers, 113.0 nanometers, 118.6 nanometers, 123.1 nanometers, 203.5 nanometers, 192.6 nanometers, 196.0 nanometers, 204.4 nanometers, 192.8 nanometers, 175.0 nanometers, 177.5 nanometers, and so on.

[0192] The disulfide bond-containing nucleic acid-lipid particles can be administered alone or in combination with a pharmaceutical carrier.

[0193] The disulfide bond-containing nucleic acid-lipid particles can be administered directly at the target site or at an administration site away from the target site.

[0194] Another aspect of the embodiments of the present application relates to a drug, which comprises a pharmaceutical carrier and the disulfide bond-containing nucleic acid-lipid particles as described in the present application.

[0195] The pharmaceutical carrier may include substances and / or compositions that do not cause allergic reactions or similar adverse reactions when administered to mammals such as humans.

[0196] The pharmaceutical carrier can be selected according to the route of administration and standard pharmaceutical practice. Examples of the pharmaceutical carrier may include water, physiological saline, 0.4% saline, 135 - 150 mM saline, phosphate buffer, 0.3% glycine, etc. Other examples of the pharmaceutical carrier may include glycoproteins for increasing stability, such as albumin, lipoprotein, globulin, etc.

[0197] The pharmaceutical carrier can be a solvent, dispersion medium, vehicle, coating, diluent, antibacterial agent, antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc.

[0198] The pharmaceutical carrier can be added after the formation of the disulfide bond-containing nucleic acid-lipid particles. In some embodiments, after the formation of the disulfide bond-containing nucleic acid-lipid particles, the disulfide bond-containing nucleic acid-lipid particles can be diluted into the pharmaceutical carrier.

[0199] The concentration of the disulfide bond-containing nucleic acid-lipid particles in the drug can vary widely, i.e., from less than about 0.05 wt%, equal to or at least about 2-5 wt%, to at most about 10-90 wt%, and is mainly selected according to the specific administration mode chosen, such as by fluid volume, viscosity, etc. For example, the concentration can be increased to reduce the fluid load associated with treatment.

[0200] The drug can be sterilized by conventional, well-known sterilization techniques.

[0201] The drug can contain pharmaceutically auxiliary substances as needed to approximate physiological conditions, such as pH regulators, pH buffers, tonicity regulators, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride, etc.

[0202] The drug can include lipid protectants as needed, which protect lipids from free radical and peroxidative damage during storage.

[0203] The drug can include lipophilic radical quenchers as needed, such as α-tocopherol, ferrioxamine.

[0204] The drug can contain: excipients, binders, gelatin, lubricants, lipid carriers, and / or flavoring agents.

[0205] The drug can be tablets, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, cachets, etc.

[0206] For in vivo administration, the administration can be by any means known in the art, for example, by injection, infusion, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal administration, or rectal administration. The administration can be achieved by single or divided doses. The drug can be administered parenterally, i.e., intra-articularly, intravenously, intraperitoneally, intravesically, subcutaneously, intrathecally, or intramuscularly. In some embodiments, the drug can be administered intravenously or intraperitoneally by bolus injection. In some embodiments, the drug can be administered by intramuscular injection, tail vein injection, and / or pulmonary inhalation.

[0207] In certain embodiments, the drug can be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. The drug can be formulated into aerosol preparations (i.e., they can be "atomized") alone or in combination with other suitable ingredients, so as to be administered by inhalation (e.g., intranasal or intratracheal). The aerosol preparation can be placed in a propellant that allows pressurization, such as dichlorodifluoromethane, propane, nitrogen, etc.

[0208] In some embodiments, the drug is one or more of an infectious disease vaccine, a cancer vaccine, a tumor immunotherapy drug, a messenger ribonucleic acid drug based on directly encoded protein, a protein replacement drug, and a protein inhibitor drug.

[0209] In some embodiments, the drug is one or more of a deoxyribonucleic acid drug, a messenger ribonucleic acid drug, a micro ribonucleic acid drug, a small interfering ribonucleic acid drug, an antisense oligonucleotide drug, a circular ribonucleic acid drug, a self-amplifying ribonucleic acid drug, and a nucleic acid aptamer drug.

[0210] Compared with messenger ribonucleic acid vaccines, the messenger ribonucleic acid drugs used in messenger ribonucleic acid therapy may require up to 1000 times the protein level to achieve a therapeutic effect. For messenger ribonucleic acid therapy, when used for messenger ribonucleic acid drugs, the disulfide bond-containing nucleic acid-lipid particles can produce a higher protein expression level and reach the therapeutic threshold faster than traditional lipid nanoparticles.

[0211] In some embodiments, the drug is a novel coronavirus vaccine.

[0212] In some embodiments, the drug is a humoral immune drug.

[0213] In some embodiments, the drug is a messenger ribonucleic acid vaccine.

[0214] Messenger ribonucleic acid vaccines usually require a relatively low protein level for production. The human immune system can amplify the immune signal through cell- and antibody-mediated immune responses. The more efficient cytoplasmic delivery property of the disulfide bond-containing nucleic acid-lipid particles compared to traditional lipid nanoparticles can help ensure the protein expression effect of messenger ribonucleic acid in the application of messenger ribonucleic acid vaccines, and can reduce the dosage, thereby reducing the toxic and side effects caused by lipids in the body and / or the inflammatory response caused by messenger ribonucleic acid.

[0215] Another aspect of the embodiments of the present application relates to the use of the disulfide bond-containing nucleic acid-lipid particles described in the present application in the preparation of a product for introducing nucleic acid into cells.

[0216] In some embodiments, the product can be a drug comprising the disulfide bond-containing nucleic acid-lipid particles described in the present application. The preparation, composition, etc. of the product can refer to the relevant descriptions of the preparation, composition, etc. of the drug comprising the disulfide bond-containing nucleic acid-lipid particles described in the present application above.

[0217] In some embodiments, when nucleic acid needs to be introduced into cells, the product comprising the disulfide bond-containing nucleic acid-lipid particles described in the present application can be contacted with the cells.

[0218] In some embodiments, the cells are mammalian cells.

[0219] Unless otherwise specifically stated, the mammals involved in this application may include, for example, primates (e.g., humans, chimpanzees, and other non-human primates), canines, felines, equines, bovines, sheep, goats, rodents (e.g., hamsters, guinea pigs, rats, and mice), lagomorphs, and domestic animals (pigs), etc.

[0220] Unless otherwise specifically stated, the cells involved in this application may include, for example, hematopoietic progenitor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or non-cycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, human embryonic kidney cells (293T cells), osteocytes, etc.

[0221] Unless otherwise specifically stated, the cells involved in this application may include, for example, cancer cells, such as lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendiceal cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, kidney cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells, etc.

[0222] Another aspect of the embodiments of this application relates to the use of the disulfide bond-containing nucleic acid-lipid particles described in this application in the preparation of a product for in vivo delivery of nucleic acids to be administered to mammals.

[0223] In some embodiments, the product may be a drug comprising the disulfide bond-containing nucleic acid-lipid particles described in this application. The preparation, composition, etc. of the product may refer to the relevant descriptions of the preparation, composition, etc. of the drug comprising the disulfide bond-containing nucleic acid-lipid particles described in this application above.

[0224] Unless otherwise specifically stated, the administrations involved in this application may include, but are not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

[0225] Unless otherwise specifically stated, the delivery involved in this application can be carried out in any manner known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal.

[0226] The term "delivery", unless otherwise specifically stated, may refer to systemic delivery that results in widespread biodistribution of the nucleic acid in a living organism, which can expose an effective amount, such as a therapeutic amount, of the nucleic acid to most parts of the body and prevent the nucleic acid from being rapidly degraded or cleared before reaching the disease site far from the administration site, such as not being degraded or cleared by first-pass organs (liver, lung, etc.) or non-specific cells. Systemic delivery for in vivo treatment can, for example, deliver therapeutic nucleic acids to target cells through body systems such as the circulatory system.

[0227] The term "delivery", unless otherwise specifically stated, may also refer to local delivery that directly delivers the nucleic acid to a target site in a living organism. For example, the disulfide bond-containing nucleic acid-lipid particles can be locally delivered by direct injection into a disease site such as a tumor, other target sites such as an inflammation site, or a target organ such as the liver, heart, pancreas, kidney, etc.

[0228] Another aspect of the embodiments of the present application relates to the use of the disulfide bond-containing nucleic acid-lipid particles described in the present application in the preparation of a drug for administration to a mammal.

[0229] The preparation, composition, etc. of the drug for administration to a mammal can refer to the relevant descriptions of the preparation, composition, etc. of the above-mentioned drug.

[0230] In some embodiments, the drug is one or more of an infectious disease vaccine, a cancer vaccine, a tumor immunotherapy drug, a messenger ribonucleic acid drug based on direct encoding of a protein, a protein replacement drug, and a protein inhibition drug.

[0231] In some embodiments, the drug is one or more of a deoxyribonucleic acid drug, a messenger ribonucleic acid drug, a micro ribonucleic acid drug, a small interfering ribonucleic acid drug, an antisense oligonucleotide drug, a circular ribonucleic acid drug, a self-amplifying ribonucleic acid drug, and a nucleic acid aptamer drug.

[0232] In some embodiments, the drug is a novel coronavirus vaccine.

[0233] In some embodiments, the drug is a humoral immune drug.

[0234] In some embodiments, the drug is a messenger ribonucleic acid vaccine.

[0235] Another aspect of the embodiments of the present application relates to the disulfide bond-containing nucleic acid-lipid particles prepared by the following preparation method, which includes:

[0236] Dissolve a cationic lipid, cholesterol or its derivative, a phospholipid, a polyethylene glycolated lipid, and a disulfide bond-containing lipid in an organic solvent to obtain an organic phase lipid solution;

[0237] Dissolve the nucleic acid in an acidic buffer solution to obtain an aqueous-phase nucleic acid solution; and

[0238] Mix the organic-phase lipid solution with the aqueous-phase nucleic acid drug solution to obtain the disulfide bond-containing nucleic acid-lipid particles.

[0239] Another aspect of the embodiments of the present application relates to a method for preparing the disulfide bond-containing nucleic acid-lipid particles described in the present application, which includes:

[0240] Dissolve the cationic lipid, the cholesterol or its derivative, the phospholipid, the polyethylene glycolated lipid, and the disulfide bond-containing lipid in an organic solvent to obtain an organic-phase lipid solution;

[0241] Dissolve the nucleic acid in an acidic buffer solution to obtain an aqueous-phase nucleic acid solution; and

[0242] Mix the organic-phase lipid solution with the aqueous-phase nucleic acid drug solution to obtain the disulfide bond-containing nucleic acid-lipid particles.

[0243] In some embodiments, the organic-phase lipid solution and the aqueous-phase nucleic acid drug solution are mixed by microfluidics, and the total flow rate ranges from 1 mL / min to 30 mL / min. Thus, it is beneficial to control the size and uniformity of the disulfide bond-containing nucleic acid-lipid particles.

[0244] In some embodiments, the organic-phase lipid solution and the aqueous-phase nucleic acid drug solution are mixed with a pipette.

[0245] In some embodiments, the preparation method includes purifying after mixing the organic-phase lipid solution with the aqueous-phase nucleic acid drug solution to obtain the disulfide bond-containing nucleic acid-lipid particles.

[0246] In some embodiments, the preparation method includes modifying a disulfide functional group on a raw lipid to obtain the disulfide bond-containing lipid.

[0247] Another aspect of the embodiments of the present application relates to a method for preventing and treating diseases, which includes administering the drug described in the present invention to a mammal.

[0248] Unless otherwise specifically stated, the administration involved in the present application may include, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

[0249] Another aspect of the embodiments of the present application relates to a method for antiviral and / or cell infection, which includes administering the drug described in the present invention to a mammal.

[0250] Another aspect of the embodiments of the present application relates to a method for introducing nucleic acid into cells, which comprises: contacting the cells with the disulfide bond-containing nucleic acid-lipid particles described in the present application.

[0251] The method of introducing the nucleic acid into the cells can be carried out in vitro or in vivo by the following steps: First, form the disulfide bond-containing nucleic acid-lipid particles as described in the present application, and then contact the disulfide bond-containing nucleic acid-lipid particles with the cells for a period of time sufficient to effect the delivery of the nucleic acid to the cells.

[0252] For in vitro applications, the nucleic acid can be delivered to any cells growing in culture, regardless of plant or animal origin, vertebrate or invertebrate, and any tissue or type. In a preferred embodiment, the cells can be animal cells, such as mammalian cells, for example human cells.

[0253] The contact between the cells and the disulfide bond-containing nucleic acid-lipid particles, when carried out in vitro, can occur in a biocompatible medium.

[0254] Another aspect of the embodiments of the present application relates to a method for introducing nucleic acid into cells, which comprises: contacting the cells with the disulfide bond-containing nucleic acid-lipid particles described in the present application in vitro.

[0255] Another aspect of the embodiments of the present application relates to a method for in vivo delivery of nucleic acid, which comprises administering the disulfide bond-containing nucleic acid-lipid particles described in the present application to a mammal.

[0256] Unless otherwise specifically stated, the administration involved in the present application can include, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

[0257] Another aspect of the embodiments of the present application relates to a method for using the disulfide bond-containing nucleic acid-lipid particles described in the present application for in vivo delivery of nucleic acid, which comprises administering the disulfide bond-containing nucleic acid-lipid particles to a mammal.

[0258] Another aspect of the embodiments of the present application relates to a method for treating a disease or disorder in a mammal, which comprises administering an effective therapeutic dose of the disulfide bond-containing nucleic acid-lipid particles described in the present application to the mammal.

[0259] The effective therapeutic dose can be an amount sufficient to produce the desired therapeutic effect.

[0260] Another aspect of the embodiments of the present application relates to a method for using the disulfide bond-containing nucleic acid-lipid particles described in the present application for treating a disease or disorder in a mammal, which comprises administering an effective therapeutic dose of the disulfide bond-containing nucleic acid-lipid particles to the mammal.

[0261] In some embodiments, the disease or disorder is selected from viral infections and tumors.

[0262] Another aspect of the embodiments of the present application relates to the use of the disulfide bond-containing nucleic acid-lipid particles described in the present application in gene therapy.

[0263] In some embodiments, the gene therapy includes one or more of deoxyribonucleic acid therapy, messenger ribonucleic acid therapy, micro ribonucleic acid therapy, small interfering ribonucleic acid therapy, antisense oligonucleotide therapy, circular ribonucleic acid therapy, self-amplifying ribonucleic acid therapy, and nucleic acid aptamer therapy.

[0264] The present invention will be further illustrated below with experimental examples. The experimental examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention in any way.

[0265] Unless otherwise specified, the process methods involved in the present application are all conventional process methods. Unless otherwise specified, the materials, reagents, etc. involved in the present application are materials and reagents that can be obtained commercially. During the experiments conducted by the inventors, lipid reagents and nucleic acid reagents from different manufacturers were tried, and it was found that on the premise that the reagents had no quality problems, the change of reagents had almost no impact on the experimental results.

[0266] Example 1: Preparation and Characterization Analysis of Nucleic Acid-Lipid Particles

[0267] 1-1. Preparation of Nucleic Acid-Lipid Particles

[0268] Information such as the type, English abbreviation, and Chinese name of each lipid used in the preparation process of nucleic acid-lipid particles is shown in Table 1 below.

[0269]

[0270] The structural formulas of the three disulfide bond-containing lipids in Table 1 are shown in Table 2 below:

[0271]

[0272] Information such as the name and sequence of the nucleic acid used in the preparation process of nucleic acid-lipid particles is listed in Table 3 below.

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279] Table 4 below lists the corresponding example numbers, English aliases, component 1 (cationic lipid), component 2 (cholesterol), component 3 (phospholipid), component 4 (PEGylated lipid), component 5 (lipid containing disulfide bond), nucleic acid, molar percentages of lipid components, and other relevant information for each example involving nucleic acid-lipid particles.

[0280]

[0281]

[0282]

[0283]

[0284]

[0285] As shown in Table 4 above, the cationic lipids involved in each nucleic acid-lipid particle are Dlin-MC3-DMA, DOTAP, SM-102, and ALC-0315, the cholesterol (Cholesterol) or its derivative is cholesterol (CHOL), the phospholipids are DSPC and DOPE respectively, the PEGylated lipids are DMG-PEG2K and ALC-0159 respectively, and the lipids containing disulfide bonds are S-DOPE, S-CHOL, and S-DSPEG-PEG2K respectively.

[0286] The molar percentages of the cationic lipid, cholesterol or its derivative, phospholipid, polyethylene glycolated lipid, and disulfide bond-containing lipid involved in each nucleic acid-lipid particle vary within the ranges of 10%-70%, 5%-65%, 0%-50%, 0%-20%, and 0%-60% respectively relative to all lipids. The nucleic acid-lipid particle with a molar percentage of 0% for the disulfide bond-containing lipid is a control nucleic acid-lipid particle, such as LNP-A, LNP-B, BNT162b2-LNP, mRNA-1273-LNP, LNP-D, LNP-E, LNP-F. The nucleic acid-lipid particle with a non-zero molar percentage of the disulfide bond-containing lipid is a disulfide bond-containing nucleic acid-lipid particle. Some nucleic acid-lipid particles differ only in nucleic acid from each other, while the lipid components and their molar percentages are the same. For example, the only difference between LNP-D and LNP-A, and between SLNP-D and SLNP-A9 is the nucleic acid. The molar percentage of phospholipid used in some nucleic acid-lipid particles is 0, such as SLNP-A1 to SLNP-A5, or the molar percentage of polyethylene glycolated lipid is 0, such as LNP-B, SLNP-B1 to SLNP-B4, SLNP-C1 to SLNP-C3.

[0287] The nucleic acids involved in each nucleic acid-lipid particle are Green Fluorescent Protein Messenger Ribonucleic Acid (GFP mRNA), Firefly Luciferase Messenger Ribonucleic Acid (FLuc mRNA), and SARS-CoV-2 S Protein Messenger Ribonucleic Acid (SP mRNA) respectively.

[0288] When preparing each nucleic acid-lipid particle, the corresponding molar percentages of component 1 (cationic lipid), component 2 (cholesterol), component 3 (phospholipid), component 4 (polyethylene glycolated lipid), and component 5 (disulfide bond-containing lipid) were dissolved in an ethanol solvent respectively according to Table 4 above to obtain the corresponding organic phase lipid solution. For example, for SLNP-A9, the molar percentages of Dlin-MC3-DMA, CHOL, DSPC, DMG-PEG2K, and S-DOPE are 41.7%, 32.1%, 8.3%, 1.3%, and 16.6% respectively.

[0289] Subsequently, according to a certain volume ratio of each organic phase lipid solution to the corresponding aqueous phase nucleic acid solution (for SLNP-A9, it can be 1:3), and a certain ratio of the total mass of all lipids in each organic phase lipid solution to the nucleic acid mass in the corresponding aqueous phase nucleic acid solution (for SLNP-A9, it can be 40:1), each nucleic acid (for SLNP-A9, GFP mRNA) was dissolved in an acidic buffer solution (for SLNP-A9, a 50 mM citric acid acidic buffer solution with a pH of 4) to obtain the corresponding aqueous phase nucleic acid solution.

[0290] Next, the organic phase lipid solution was mixed with the aqueous phase nucleic acid drug solution. For example, for SLNP-A9, through the rapid nucleic acid nanodrug preparation system (INano TM L) of Myanna (Shanghai) Instrument Technology Co., Ltd., under the microfluidic mixing conditions where the flow rate ratio of the organic phase lipid solution to the aqueous phase nucleic acid solution is 1:3 and the total flow rate is 12 mL / min, the two phases were converged and mixed in the chip to obtain a slightly white solution.

[0291] Finally, purification was carried out to obtain each nucleic acid-lipid particle containing a disulfide bond (such as SLNP-A9) and each control nucleic acid-lipid particle with a molar percentage of disulfide bond-containing lipid of 0% (such as LNP-A). For example, the obtained slightly white solution can be diluted with 10 times the volume of a 10 mM PBS buffer solution with a pH of 7 and ultrafiltered through an ultrafiltration tube with a 30 KD membrane pore size to remove ethanol and purify, thereby obtaining SLNP-A9.

[0292] 1-2. Characterization and analysis of nucleic acid-lipid particles

[0293] To characterize each nucleic acid-lipid particle prepared with different lipid formulations, different lipid molar percentages, and different nucleic acids, dynamic light scattering (DLS) was used to detect the particle size and polydispersity index (PDI) of some nucleic acid-lipid particles, and nano-flow cytometry (nFCM) was used to detect the encapsulation efficiency. The physical properties such as the particle size, PDI, and encapsulation efficiency of each nucleic acid-lipid particle are summarized in Table 5 below. The data in Table 5 show that when the molar percentages of cationic lipid, cholesterol or its derivatives, phospholipid, polyethylene glycolated lipid, and disulfide bond-containing lipid are in the ranges of 10%-70%, 5%-65%, 0%-50%, 0%-20%, and 0%-60% respectively, both the control nucleic acid-lipid particles and the nucleic acid-lipid particles containing disulfide bonds were successfully prepared, and the physical properties are within an acceptable range.

[0294]

[0295] Transmission Electron Microscope (TEM) and Cryo-Electron Microscopy (Cryo-EM) were used to observe the morphological characteristics of nucleic acid-lipid particles. The morphological images of SLNP-A9 under TEM and Cryo-EM are shown in Figure 1 .

[0296] from Figure 1 It can be seen that SLNP-A9 is in the shape of a uniformly dispersed sphere, and the actual particle size is about 100 nm, which is slightly smaller than the hydrodynamic radius (118.6 nm) detected by DLS in Table 5 above, and the two can confirm each other.

[0297] This example shows that nucleic acid-lipid particles containing disulfide bonds can be successfully prepared within a reasonable range of component ratios, for example, nucleic acid-lipid particles containing disulfide bonds can be successfully prepared when the molar percentages of cationic lipids, cholesterol or its derivatives, phospholipids, pegylated lipids and disulfide bond-containing lipids are in the ranges of 10%-70%, 5%-65%, 0%-50%, 0%-20%, and 0.5%-60%, respectively.

[0298] This example also illustrates that disulfide bond-containing lipids can be used to prepare disulfide bond-containing nucleic acid-lipid particles with different lipid formulations, different lipid ratios, and different nucleic acids.

[0299] Furthermore, the successful preparation of BNT162b2-SLNP and mRNA-1273-SLNP also demonstrates that the method of incorporating disulfide bond-containing lipids into the basic nucleic acid-lipid nanoparticle (LNP) system (such as the commercial lipid formulation of BNT162b2 new coronavirus vaccine and the commercial lipid formulation of mRNA-1273 new coronavirus vaccine) is feasible and universal.

[0300] Example 2: In vitro experiments with nucleic acid-lipid particles

[0301] 2-1. Transfection of nucleic acid-lipid particles prepared with different formulation ratios in human embryonic kidney cells (293T cells)

[0302] According to 1.5×10 per well 4 293T cells were seeded into 96-well plates.

[0303] The 96-well plate was placed at 37°C and 5% CO 2 Incubate in an incubator for 24 hours.

[0304] The control nucleic acid-lipid particle LNP-A and the disulfide bond-containing nucleic acid-lipid particles SLNP-A6, SLNP-A7, SLNP-A9, and SLNP-A11 were directly diluted in Dulbecco's modified Eagle's medium (DMEM complete medium) at doses of 50 ng, 100 ng, and 150 ng of GFP mRNA, respectively, and the 293T cells in the 96-well plate were transfected separately.

[0305] After 6 - 24 hours, the transfection of the cells was observed by fluorescence microscopy imaging and flow cytometry analysis and statistics, respectively. The green fluorescent protein microscope images and flow analysis and statistics data obtained are shown in Figure 2 .

[0306] From Figure 2 It can be observed that at the same GFP mRNA dose, compared with the control nucleic acid-lipid particle LNP-A without disulfide bond-containing lipids, the disulfide bond-containing nucleic acid-lipid particles SLNP-A6, SLNP-A7, SLNP-A9, and SLNP-A11 promoted the entry of GFP mRNA into the cytoplasm and translation into protein to a greater extent, indicating that the transfected 293T cells expressed more green fluorescent protein, verifying the more efficient cytoplasmic delivery of the disulfide bond-containing nucleic acid-lipid particles at the cellular level.

[0307] At the same GFP mRNA dose, for the disulfide bond-containing nucleic acid-lipid particles SLNP-A6, SLNP-A7, SLNP-A9, and SLNP-A11, as the molar percentage of the disulfide bond-containing lipid S-DOPE increased, their ability to express green fluorescent protein also generally increased gradually, the fluorescence signal in the 293T cells generally increased gradually, and the expression effect of SLNP-A9 was the best.

[0308] Figure 2 As can be seen in

[0309] In addition, regarding the commercial lipid formulations of the BNT162b2 SARS-CoV-2 vaccine (ALC-0315:CHOL:DSPC:ALC-0159 = 46.3:42.7:9.4:1.6) and the mRNA-1273 SARS-CoV-2 vaccine (SM-102:CHOL:DSPC:DMG-PEG2K = 50:38.5:10:1.5), and the control nucleic acid-lipid particles BNT162b2-LNP and mRNA-1273-LNP of GFP mRNA, as well as the disulfide-containing nucleic acid-lipid particles BNT162b2-SLNP (ALC-0315:CHOL:DSPC:ALC-0159:S-DOPE = 38.7:35.6:7.8:1.3:16.6) and mRNA-1273-SLNP (SM-102:CHOL:DSPC:DMG-PEG2K:S-DOPE = 41.7:32.1:8.3:1.3:16.6) incorporating the disulfide-containing lipid S-DOPE, 293T cells were transfected with BNT162b2-LNP, BNT162b2-SLNP, mRNA-1273-LNP, and mRNA-1273-SLNP at four GFP mRNA doses of 0 ng (Blank), 50 ng, 100 ng, and 150 ng using the above methods respectively. Flow cytometry analysis data of the GFP fluorescence intensity in 293T cells after transfection are as Figure 3 shown.

[0310] According to Figure 3 the results of the flow cytometry experiments in

[0311] Figure 3 it was observed that, at the same GFP mRNA dose, compared with the control nucleic acid-lipid particles BNT162b2-LNP and mRNA-1273-LNP, the cells transfected with the disulfide-containing nucleic acid-lipid particles BNT162b2-SLNP and mRNA-1273-SLNP showed stronger fluorescence signals, indicating that the disulfide-containing nucleic acid-lipid particles based on the commercial lipid formulations of the Pfizer BNT162b2 SARS-CoV-2 vaccine and the Moderna mRNA-1273 SARS-CoV-2 vaccine also improved the transfection ability of GFP mRNA.

[0312] This example also shows that disulfide-containing lipids can be used for the preparation of nucleic acid-lipid particles containing disulfide bonds with different lipid formulations and different lipid ratios. The method of incorporating disulfide-containing lipids into the basic nucleic acid-lipid nanoparticle (Lipid Nanoparticles, LNP) system is universal. The scheme provided by the embodiments of the present invention is applicable to different nucleic acid-lipid nanoparticle formulations.

[0313] 2-2. Cellular Uptake and Mechanism of Nucleic Acid-Lipid Particles

[0314] To explore the pathway by which nucleic acid-lipid particles are taken up by cells, Cy5-labeled GFP mRNA was used as a model to track the distribution of nucleic acid-lipid particles at the subcellular level.

[0315] 293T cells were incubated with disulfide-containing nucleic acid-lipid particles SLNP-D (Dlin-MC3-DMA:CHOL:DSPC:DMG-PEG2K:S-DOPE = 41.7:32.1:8.3:1.3:16.6; Cy5-GFP mRNA), naked Cy5-GFP mRNA without encapsulation (Naked mRNA), and control nucleic acid-lipid particles LNP-D without disulfide bonds (Dlin-MC3-DMA:CHOL:DSPC:DMG-PEG2K:S-DOPE = 50:38.5:10:1.5:0; Cy5-GFP mRNA) for two different times, 2 hours (2h) and 4 hours (4h), respectively. Then, the cell nuclei and lysosomes were stained with Hoechst 33342 (H33342) nuclear dye and LysoTracker TM Green (Lysotracker) lysosomal green fluorescent probe, respectively. After that, confocal imaging and flow analysis were performed. The obtained confocal images and flow analysis data are shown in Figure 4 .

[0316] As Figure 4 (a) shown in the confocal images, most of the red fluorescence of the disulfide-containing nucleic acid-lipid particles SLNP-D is distributed in the cytoplasm, and only a small part overlaps with the green fluorescence of lysosomes, which means that few disulfide-containing nucleic acid-lipid particles SLNP-D are captured by lysosomes, and the disulfide-containing nucleic acid-lipid particles SLNP-D can efficiently deliver to the cytoplasm. In addition, cells incubated with disulfide-containing nucleic acid-lipid particles SLNP-D showed more obvious red fluorescence signals. However, naked Cy5-GFP mRNA without encapsulation and control nucleic acid-lipid particles LNP-D without disulfide bonds had only weak fluorescence signals.

[0317] Figure 4(b) Flow cytometry analysis results are consistent with Figure 4 (a) The confocal results. Compared with the control nucleic acid-lipid particle LNP-D, the number of disulfide bond-containing nucleic acid-lipid particles SLNP-D loaded with Cy5-GFP mRNA taken up by 293T cells was significantly increased.

[0318] The above results indicate that compared with naked Cy5-GFP mRNA and the control nucleic acid-lipid particle LNP-D, the disulfide bond-containing nucleic acid-lipid particle SLNP-D can be taken up by cells more efficiently and can deliver to the cytoplasm more efficiently.

[0319] To further explore the mechanism of disulfide bond-containing nucleic acid-lipid particles entering cells, cell uptake studies were carried out using the following different inhibitors.

[0320] Among them, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) inhibits thiol-mediated cell uptake by blocking the thiols on the outer surface of the cell membrane.

[0321] Chlorpromazine (CPZ) is a cationic amphiphilic drug that can inhibit clathrin-mediated endocytosis by causing clathrin and its adaptor proteins to migrate from the plasma membrane to endosomes, thus inhibiting the formation of clathrin-coated vesicles.

[0322] Wortmannin can inhibit macropinocytosis by inhibiting phosphatidylinositol-3-phosphate (PI3K).

[0323] Methyl-beta-cyclodextrin (m-βCD) is a cyclic oligomer of pyranosides that can inhibit caveolin-mediated endocytosis.

[0324] First, each 293T cell was pre-incubated with DMEM culture medium containing the above inhibitors (DTNB, CPZ, Wortmannin, m-βCD) for 2 hours. At the same time, 293T cells were pre-incubated with DMEM culture medium without the above inhibitors for 2 hours as a control group (No inhibitor).

[0325] Subsequently, DMEM culture medium containing disulfide bond-containing nucleic acid-lipid particle SLNP-A9 was added, and continued to co-incubate with the aforementioned 293T cells respectively.

[0326] After 24 hours, flow cytometry analysis was performed on the GFP protein expression of each 293T cell, and the results are shown in Figure 5 .

[0327] According to Figure 5 Figure 5 Results showed that the expression levels of GFP protein in 293T cells in the m-βCD group and the Wortmannin group were close to those in the No inhibitor control group, and the expression level of GFP protein in 293T cells in the CPZ group only decreased slightly, indicating that the inhibitors m-βCD, Wortmannin, and CPZ had no significant effect on the cellular uptake efficiency of the disulfide-containing nucleic acid-lipid particle SLNP-A9.

[0328]

[0328] In contrast, after adding the thiol exchange inhibitor DTNB, the expression of the disulfide-containing nucleic acid-lipid particle SLNP-A9 encapsulating GFP mRNA in cells decreased significantly. The expression level of GFP protein in cells decreased significantly, indicating that the inhibitor DTNB blocked the thiols on the cell membrane surface, thereby significantly inhibiting the cellular uptake of the disulfide-containing nucleic acid-lipid particle SLNP-A9. Therefore, the cellular uptake mode of thiol exchange is the key to achieving efficient cytoplasmic delivery of disulfide-containing nucleic acid-lipid particles.

[0329]

[0329] The above results indicate that the disulfide-containing nucleic acid-lipid particles mainly enter cells through the thiol-mediated cellular uptake mechanism, and also show that the disulfide-containing nucleic acid-lipid particles can be efficiently taken up by cells through the thiol-mediated cellular uptake mechanism.

[0330] 2-3. The disulfide-containing lipids and disulfide-containing nucleic acid-lipid particles have low cytotoxicity

[0331]

[0331] The Cell Counting Kit-8 (CCK-8) assay was used to evaluate the cytotoxicity of various concentrations of the disulfide-containing lipid S-DOPE and various nucleic acid-lipid particles LNP-A, SLNP-A6 to SLNP-A11 on cells.

[0332]

[0332] On the one hand, the disulfide-containing lipid S-DOPE with concentrations of 0 μM (Blank), 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM were added to some 293T cells, respectively.

[0333]

[0333] On the other hand, the nucleic acid-lipid particles LNP-A, SLNP-A6 to SLNP-A11 prepared with different molar percentages of S-DOPE were added to other 293T cells, respectively. The final concentration of GFP mRNA in each nucleic acid-lipid particle was 1 μg / ml.

[0334]

[0334] Meanwhile, Lipofectamine TM 3000 (Lipo3000) with a final concentration of GFP mRNA also being 1 μg / ml was used as a positive control to treat 293T cells.

[0335] After 24 hours of transfection, the CCK-8 kit was used to label the cells, and a microplate reader was used to analyze the cell viability. The results are as Figure 6 shown.

[0336] See Figure 6 the left part of . Combining the data of the control group Blank with a concentration of 0 μM of the disulfide bond-containing lipid S-DOPE, the disulfide bond-containing lipid S-DOPE had almost no toxicity to the cells during the process of the concentration increasing from 1 μM to as high as 30 μM.

[0337] In addition, see Figure 6 the right part of . Combining the data of the control group Blank without nucleic acid-lipid particles and the control nucleic acid-lipid particle LNP-A without disulfide bonds, as the proportion of S-DOPE increased, after the corresponding disulfide bond-containing nucleic acid-lipid particles were added to the cells, the cell survival rate was above 90%, showing very low cytotoxicity. In contrast, the cell viability of the Lipofectamine TM 3000 group was significantly lower.

[0338] The above data indicate that the disulfide bond-containing lipid S-DOPE itself has low cytotoxicity, and the disulfide bond-containing nucleic acid-lipid particles also have low cytotoxicity.

[0339] 2-4. Antigen expression of severe acute respiratory syndrome coronavirus 2 spike protein mRNA (SP mRNA) encapsulated in disulfide bond-containing nucleic acid-lipid particles on 293T cells

[0340] The efficient cytoplasmic delivery and low / no cytotoxicity of the disulfide bond-containing nucleic acid-lipid particles can facilitate the further application of the disulfide bond-containing nucleic acid-lipid particles in gene therapy at the cellular level.

[0341] The disulfide bond-containing nucleic acid-lipid particles SLNP-E encapsulating SP mRNA and the control nucleic acid-lipid particle LNP-E were transfected into 293T cells respectively.

[0342] Subsequently, the enzyme-linked immunosorbent assay (ELISA) method was used to measure the antigen expression level in the cell supernatant after 24 hours of transfection. The obtained concentration data of the S protein antigen are as Figure 7 shown.

[0343] See Figure 7, compared with the control nucleic acid-lipid particle LNP-E without disulfide bonds, the antigen expression level of the nucleic acid-lipid particle SLNP-E containing disulfide bonds is about 10 times higher. The nucleic acid-lipid particle SLNP-E containing disulfide bonds and loaded with SP mRNA expressed more antigen proteins in 293T cells, further demonstrating that the nucleic acid-lipid particle containing disulfide bonds has higher cytoplasmic delivery efficiency.

[0344] The above in vitro experimental studies confirmed that the nucleic acid-lipid particle containing disulfide bonds has an efficient cytoplasmic delivery advantage at the cellular level.

[0345] Example 3. In vivo transfection experiment of nucleic acid-lipid particles

[0346] To further characterize the in vivo transfection activity of nucleic acid-lipid particles, three 8-week-old female Balb / c mice were intramuscularly injected with PBS buffer (PBS), control nucleic acid-lipid particles (LNP-F), and nucleic acid-lipid particles containing disulfide bonds (SLNP-F) at a dose of 2 μg FLuc mRNA per mouse.

[0347] At 1 hour (1h), 2 hours (2h), 3 hours (3h), 4 hours (4h), 5 hours (5h), 6 hours (6h), 12 hours (12h), and 24 hours (24h) after injection, potassium D-luciferin was intraperitoneally injected into the mice at a dose of 150 mg / kg, and then the three mice were subjected to in vivo bioluminescence imaging using a small animal in vivo fluorescence imaging system (PerkinElmer, model: Lumina XRMSIII), and the obtained images and fluorescence data statistical charts are shown in Figure 8 (a) and Figure 8 (b).

[0348] As shown in Figure 8 (a) and Figure 8 (b), compared with the control nucleic acid-lipid particle LNP-F loaded with FLuc mRNA, the nucleic acid-lipid particle SLNP-F containing disulfide bonds and loaded with FLuc mRNA can express more firefly luciferase proteins in live mice, thus generating a stronger fluorescence signal. The luciferase expression of the nucleic acid-lipid particle SLNP-F containing disulfide bonds and loaded with FLuc mRNA reached its peak at 3 hours, and its luminescence intensity was 3 times that of the control nucleic acid-lipid particle LNP-F loaded with FLuc mRNA.

[0349] This example verified the efficient cytoplasmic delivery of the nucleic acid-lipid particle containing disulfide bonds at the in vivo level, indicating that the nucleic acid-lipid particle containing disulfide bonds also has excellent cytoplasmic delivery effects at the in vivo level.

[0350] In addition, to investigate the in vivo transfection activity of nucleic acid-lipid particles administered through different routes of administration, six 8-week-old mice were respectively administered PBS buffer (PBS), control nucleic acid-lipid particles (LNP-F), or nucleic acid-lipid particles containing disulfide bonds (SLNP-F) by tail vein injection or pulmonary inhalation at a dose of 5 μg FLuc mRNA per mouse.

[0351] Three hours after injection, potassium D-luciferin was intraperitoneally injected into each mouse at a dose of 150 mg / kg, and then in vivo bioluminescence imaging was performed on the six mice using a small animal in vivo fluorescence imaging system (PerkinElmer / Lumina XRMSIII), and the obtained images are respectively shown in (a) and Figure 9 (a) and Figure 9 (b).

[0352] As shown in Figure 9 (a) and Figure 9 (b), regardless of whether it is by tail vein injection or pulmonary inhalation, compared with the control nucleic acid-lipid particles LNP-F loaded with FLuc mRNA, the nucleic acid-lipid particles SLNP-F containing disulfide bonds and loaded with FLuc mRNA can express more firefly luciferase proteins in live mice, thus generating stronger fluorescence signals.

[0353] This example verifies the efficient cytoplasmic delivery of nucleic acid-lipid particles containing disulfide bonds at the in vivo level through different routes of administration, indicating that nucleic acid-lipid particles containing disulfide bonds have excellent cytoplasmic delivery effects in various routes of administration.

[0354] Example 4. Application of nucleic acid-lipid particles containing disulfide bonds in a novel coronavirus model

[0355] On day 0, control nucleic acid-lipid particles LNP-E containing 2 μg SP mRNA and nucleic acid-lipid particles SLNP-E containing disulfide bonds were intramuscularly injected into mice for the first immunization. The sera of the mice after the first immunization were collected on day 14. A booster injection was given on day 15 for a second immunization, and the sera of the mice after the second immunization were collected on day 30.

[0356] Subsequently, the titers of IgG antibodies related to the S protein of the novel coronavirus (anti-S protein IgG titer) in the sera of the mice after the first and second immunizations were respectively measured using the ELISA method, and the results are as shown in Figure 10 shown.

[0357] From Figure 10It can be seen that, compared with the control nucleic acid-lipid particle LNP-E without disulfide bonds, the nucleic acid-lipid particle SLNP-E containing disulfide bonds induced a higher level of binding antibody expression after immunization, generating a higher antibody level, indicating that the humoral immunity was enhanced.

[0358] This example shows that the novel coronavirus messenger ribonucleic acid vaccine based on the nucleic acid-lipid particle containing disulfide bonds has an enhancing effect on the humoral immune response, demonstrating the application prospects of the nucleic acid-lipid particle containing disulfide bonds.

[0359] Example 5. Biosafety of the nucleic acid-lipid particle containing disulfide bonds

[0360] Vaccines of PBS buffer (PBS), low-dose (2 μg SP mRNA) nucleic acid-lipid particle SLNP-E containing disulfide bonds, and high-dose (10 μg SP mRNA) nucleic acid-lipid particle SLNP-E containing disulfide bonds were administered to mice by intramuscular injection. Whole blood was collected 48 hours after injection and serum was separated by centrifugation.

[0361] Subsequently, a kit was used to detect liver function indicators (aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB)) and kidney function indicators (urea (UREA), creatinine (CREA), uric acid (UA)). The obtained data are shown in Figure 11 .

[0362] As Figure 11 shown, the indicators corresponding to the low-dose and high-dose nucleic acid-lipid particles SLNP-E containing disulfide bonds are all comparable to those of PBS and within the normal range, and it has no effect on kidney function and liver function, indicating that the nucleic acid-lipid particle SLNP-E containing disulfide bonds has good biosafety.

[0363] This example shows that the novel coronavirus messenger ribonucleic acid vaccine based on the nucleic acid-lipid particle containing disulfide bonds not only has high transfection efficiency, but also has low toxicity and good biocompatibility in vivo, and is an ideal delivery vector.

[0364] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A nucleic acid-lipid particle containing a disulfide bond, characterized in that, it comprises: a nucleic acid; a cationic lipid, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 10% to 70%; cholesterol or its derivative, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 5% to 65%; a phospholipid, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0% to 50%; a polyethylene glycolylated lipid, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0% to 20%; and a lipid containing a disulfide bond, the molar percentage of which relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 0.5% to 60%.

2. The nucleic acid-lipid particle containing a disulfide bond according to claim 1, characterized in that, the lipid containing a disulfide bond includes a terminal disulfide bond; and / or the lipid containing a disulfide bond includes one or more of a cationic lipid containing a disulfide bond, a cholesterol or its derivative containing a disulfide bond, a phospholipid containing a disulfide bond, and a polyethylene glycolylated lipid containing a disulfide bond; and / or the lipid containing a disulfide bond includes a lipid modified with a disulfide functional group; and / or the lipid containing a disulfide bond includes a lipid modified with a disulfide cyclic functional group; and / or the lipid containing a disulfide bond includes a lipid modified with 1,2-dithiolane; and / or the lipid containing a disulfide bond includes one or more of a cationic lipid modified with 1,2-dithiolane, a cholesterol or its derivative modified with 1,2-dithiolane, a phospholipid modified with 1,2-dithiolane, and a polyethylene glycolylated lipid modified with 1,2-dithiolane; and / or the lipid containing a disulfide bond includes one or more of dioleoyl phosphatidylethanolamine modified with 1,2-dithiolane, cholesterol modified with 1,2-dithiolane, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 modified with 1,2-dithiolane; and / or The disulfide bond-containing lipid includes a structural formula as follows or one or more of the compounds of; and / or the molar percentage of the lipid containing a disulfide bond relative to all lipids in the nucleic acid-lipid particle containing a disulfide bond ranges from 1% to 40%.

3. The nucleic acid-lipid particle containing a disulfide bond according to claim 1 or 2, characterized in that, the cationic lipid includes one or more of methyl 4-(N,N-dimethylamino)butyrate (dilinoleoyl), trimethyl-2,3-dioleyloxypropylammonium bromide, heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, ((4-hydroxybutyl)azaalkanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); and / or the cholesterol or its derivative includes cholesterol; and / or the phospholipid includes distearoyl phosphatidylcholine, and / or dioleoyl phosphatidylethanolamine; and / or The PEGylated lipid includes 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and / or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000.

4. The disulfide bond-containing nucleic acid-lipid particle according to claim 3, wherein, the molar percentage of the cationic lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle ranges from 25% to 60%; and / or the molar percentage of the cholesterol or its derivative relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle ranges from 20% to 45%; and / or the molar percentage of the phospholipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle ranges from 0% to 15%; and / or the molar percentage of the PEGylated lipid relative to all lipids in the disulfide bond-containing nucleic acid-lipid particle ranges from 0% to 2%.

5. The disulfide bond-containing nucleic acid-lipid particle according to claim 1 or 2, wherein, the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle ranges from 10:1 to 100:1; or the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle ranges from 20:1 to 40:1; or the mass ratio of all lipids to the nucleic acid in the disulfide bond-containing nucleic acid-lipid particle is 40:

1.

6. The disulfide bond-containing nucleic acid-lipid particle according to claim 1 or 2, wherein, the nucleic acid includes one or more of deoxyribonucleic acid, messenger ribonucleic acid, micro ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleotide, circular ribonucleic acid, self-amplifying ribonucleic acid, and nucleic acid aptamer; or the nucleic acid includes one or more of green fluorescent protein messenger ribonucleic acid, firefly luciferase messenger ribonucleic acid, and severe acute respiratory syndrome coronavirus 2 spike protein messenger ribonucleic acid.

7. The disulfide bond-containing nucleic acid-lipid particle according to claim 1 or 2, wherein, the particle size of the disulfide bond-containing nucleic acid-lipid particle ranges from 50 nanometers to 300 nanometers; or the particle size of the disulfide bond-containing nucleic acid-lipid particle ranges from 110 nanometers to 210 nanometers.

8. A drug, wherein, it comprises a pharmaceutical carrier and the disulfide bond-containing nucleic acid-lipid particle according to any one of claims 1-7.

9. The drug according to claim 8, wherein, it is one or more of an infectious disease vaccine, a cancer vaccine, a tumor immunotherapy drug, a messenger ribonucleic acid drug based on direct encoding of a protein, a protein replacement drug, and a protein inhibition drug; and / or it is one or more of a deoxyribonucleic acid drug, a messenger ribonucleic acid drug, a micro ribonucleic acid drug, a small interfering ribonucleic acid drug, an antisense oligonucleotide drug, a circular ribonucleic acid drug, a self-amplifying ribonucleic acid drug, and a nucleic acid aptamer drug.

10. The drug according to claim 8, wherein, it is a severe acute respiratory syndrome coronavirus 2 vaccine; and / or it is a humoral immune drug; and / or it is a messenger ribonucleic acid vaccine.

11. Use of the disulfide bond-containing nucleic acid-lipid particle according to any one of claims 1-7 in the preparation of a product for introducing nucleic acid into cells.

12. The use according to claim 11, wherein, the cell is a mammalian cell.

13. Use of the disulfide bond-containing nucleic acid-lipid particle according to any one of claims 1-7 in the preparation of a product for in vivo delivery of nucleic acid to be administered to a mammal.

14. Use of the disulfide bond-containing nucleic acid-lipid particle according to any one of claims 1-7 in the preparation of a drug to be administered to a mammal.

15. The use according to claim 14, wherein, the drug is one or more of an infectious disease vaccine, a cancer vaccine, a tumor immunotherapy drug, a messenger ribonucleic acid drug based on direct encoding of a protein, a protein replacement drug, and a protein inhibition drug; and / or the drug is one or more of a deoxyribonucleic acid drug, a messenger ribonucleic acid drug, a micro ribonucleic acid drug, a small interfering ribonucleic acid drug, an antisense oligonucleotide drug, a circular ribonucleic acid drug, a self-amplifying ribonucleic acid drug, and a nucleic acid aptamer drug.

16. The use according to claim 14, wherein, the drug is a novel coronavirus vaccine; and / or the drug is a humoral immune drug; and / or the drug is a messenger ribonucleic acid vaccine.

17. A method for preparing the disulfide bond-containing nucleic acid-lipid particle according to any one of claims 1-7, wherein, comprising: dissolving the cationic lipid, the cholesterol or its derivative, the phospholipid, the polyethylene glycolated lipid, and the disulfide bond-containing lipid in an organic solvent to obtain an organic phase lipid solution; dissolving the nucleic acid in an acidic buffer solution to obtain an aqueous phase nucleic acid solution; and mixing the organic phase lipid solution with the aqueous phase nucleic acid drug solution to obtain the disulfide bond-containing nucleic acid-lipid particle.

18. The preparation method according to claim 17, wherein, the organic phase lipid solution and the aqueous phase nucleic acid drug solution are mixed by microfluidics, and the total flow rate ranges from 1 mL / min to 30 mL / min; and / or the organic phase lipid solution and the aqueous phase nucleic acid drug solution are mixed with a pipette.

19. The preparation method according to claim 17, wherein, it comprises purifying after mixing the organic phase lipid solution with the aqueous phase nucleic acid drug solution to obtain the disulfide bond-containing nucleic acid-lipid particle.

20. The preparation method according to claim 17, wherein, it comprises modifying a disulfide functional group on a raw material lipid to obtain the disulfide bond-containing lipid.

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