Negative electricity modified biomineralized lipid nanoparticle as well as preparation method and application thereof

Abstract

The invention provides negatively-modified biomineralized lipid nanoparticles as well as a preparation method and application thereof. The negatively-modified biomineralized lipid nanoparticles consist of lipid nanoparticles and mineralized shells, wherein the surfaces of the lipid nanoparticles are modified with negatively-charged molecules, and the mineralized shells are deposited on the surfaces of the lipid nanoparticles. According to the particles, negative molecules can be efficiently modified on the surface of LNP, the negative molecules are effectively prevented from being wrapped in the LNP, mineralization of lipid nanoparticles is promoted, and the stability of the LNP is improved; the cell entering efficiency is improved, and the delivery capacity of the LNP on nucleic acid drugs is enhanced; meanwhile, the compound can be used as a treatment medicine to improve the curative effect of LNP, and has multiple effects.

Description

Technical Field

[0001] The present invention relates to the technical field of lipid nanoparticles, and particularly relates to a negatively charged biomineralized lipid nanoparticle, a preparation method thereof and an application thereof. Background Art

[0002] Nucleic acid drugs include various nucleic acid molecules such as deoxyribonucleic acid (DNA), antisense oligonucleotides (dASO), small interfering ribonucleic acid (siRNA), messenger ribonucleic acid (mRNA), etc., and have great application potential in the prevention and treatment of various diseases such as genetic diseases, infectious diseases, and cancers. However, the negative charge, hydrophilicity of nucleic acid drugs, and sensitivity to nuclease degradation make it difficult for them to function independently. In order to enable nucleic acid drugs to function more efficiently in cells, a number of studies have developed various delivery carriers to protect and transport nucleic acid molecules into target cells to play a role. Among them, lipid nanoparticles (LNP) are the most promising delivery platforms for clinical applications, and several siRNA and mRNA drugs using LNP as delivery carriers have been approved for marketing.

[0003] LNP is usually self-assembled by ionizable lipids, helper phospholipids, cholesterol, polyethylene glycol (PEG)-ylated lipids and nucleic acids through hydrophobic interaction and electrostatic interaction. However, the relatively weak interaction between lipid components results in poor stability of LNP. Recent studies have shown that slight external forces (dropping, repeated aspiration with a syringe, shaking and rotation) can cause LNP to aggregate and break. In addition, the relatively low phase transition temperature of lipid components results in very limited thermal stability of LNP. For example, the mRNA-LNP vaccine developed by Pfizer and BioNTech needs to be stored at -70°C, which greatly increases the transportation difficulty of the vaccine. After optimization, the mRNA-LNP vaccine developed by Moderna can only be stored at 2-8°C for about 30 days although its thermal stability has been improved. Such stringent storage conditions make it difficult for existing mRNA-LNP vaccines to be widely applied in countries with limited medical resources.

[0004] In nature, organisms can form various inorganic crystals through biomineralization, such as bones and shells, so as to provide structural support and protection for soft tissues. Inspired by the biomineralization process, a number of studies have successfully used this strategy to construct mineralized structures on the basis of biomacromolecules and nanoparticles, thereby enhancing the stability of the original structure, and are widely used as drug delivery carriers and imaging probe carriers. For example, the thermal stability of subunit vaccines after biomineralization has been significantly improved, and the storage time at room temperature has been greatly extended. Summary of the Invention

[0005] To solve the problems that existing LNPs are vulnerable to external force damage, and have poor structural and thermal stability, the present invention provides a biomineralization method for LNPs, which can form a calcium phosphate (CaP) shell on the surface of LNPs, thereby protecting the original structure of LNPs. First, the present invention modifies negatively charged molecules on the surface of LNPs through three methods (directed introduction, click ligation, and surface insertion), and then uses the negatively charged molecules to attract and bind calcium ions (Ca 2+ ) and phosphate ions (PO4 3- ), thereby inducing a biomineralization reaction and gradually forming a CaP mineralized shell on the surface of LNPs. After modifying the negatively charged molecules by the methods of click ligation and surface insertion, the cellular uptake efficiency of LNPs is further improved, and the mineralized LNPs can still maintain a high cellular uptake ability.

[0006] To achieve the above object of the present invention, on the one hand, the present invention provides a composite lipid nanoparticle having a mineralized shell. For example, the composite lipid nanoparticle is composed of a lipid nanoparticle (abbreviated as LNP) surface-modified with a negatively charged molecule and a mineralized shell deposited on the surface of the lipid nanoparticle.

[0007] In a preferred embodiment of the present invention, the thickness of the mineralized shell is 50 - 80 nm, such as 60 nm, 70 nm.

[0008] In one embodiment of the present invention, the lipid nanoparticle includes a lipid component, and the lipid component includes an ionizable lipid, a helper phospholipid, cholesterol and its derivatives, and a polyethylene glycolated lipid.

[0009] In one embodiment of the present invention, the ionizable lipid accounts for 20 mol% - 80 mol% of the lipid component in the lipid nanoparticle, such as 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%, 75 mol%;

[0010] In one embodiment of the present invention, the ionizable lipid includes, but is not limited to, (heptadec-9-yl) 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoate (SM-102), [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), methyl 4-(N,N-dimethylamino)butyrate bis(linoleate) (DLin-MC3-DMA), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12), bis((Z)-non-2-en-1-yl) 9-(4-(dimethylamino)butanoyloxy)heptanedioate (L319), N2,2-bis(linoleoyl)-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), (heptadec-9-yl) 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoate (Lipid5), 1,1′-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl)azanediyl]bis(dodecan-2-ol) (C12-200), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium bromide (DDAB), tetra(8-methylnonyl) 3,3′,3”,3”′-{[(methylazanediyl)bis(propane-3,1-diyl)]bis(azanetriyl)}tetrapropionate (306Oi10), or one or more thereof.

[0011] In one embodiment of the present invention, the auxiliary phospholipid accounts for 2 mol% to 30 mol% of the lipid component in the lipid nanoparticle, such as 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%;

[0012] In one embodiment of the present invention, the auxiliary phospholipid includes, but is not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or one or more thereof.

[0013] In one embodiment of the present invention, the cholesterol and its derivatives account for 10 mol% to 50 mol% of the lipid component in the lipid nanoparticles, such as 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%.

[0014] In one embodiment of the present invention, the cholesterol and its derivatives include, but are not limited to, one or more of β-sitosterol, cholestanol, cholestanone, cholesterol, cholestenone, 7β-hydroxy cholesterol, 7α-hydroxy cholesterol.

[0015] In one embodiment of the present invention, the polyethylene glycolylated lipid accounts for 0.3 mol% - 30 mol% of the lipid component in the lipid nanoparticles, such as 0.5 mol%, 1 mol%, 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%.

[0016] In one embodiment of the present invention, the polyethylene glycolylated lipid includes, but is not limited to, one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glycerol-3-methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG).

[0017] In one embodiment of the present invention, the lipid nanoparticles further include a nucleic acid drug, the lipid nanoparticles encapsulate the nucleic acid drug, and the nucleic acid drug includes antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, etc.

[0018] In one embodiment of the present invention, the amount of the nucleic acid drug is represented by the N / P ratio, that is, the ratio of the nitrogen atom in the ionizable lipid to the phosphate group in the nucleic acid. In the present invention, the N / P ratio of the LNP is about 4:1 to 16:1 and between 4:1 and 16:1.

[0019] In a specific embodiment of the present invention, the ionizable lipid is 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid 1-octylnonyl ester (SM-102), the auxiliary phospholipid is distearoyl phosphatidylcholine (DSPC), cholesterol, the polyethylene glycolated lipid is dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000), and the nucleic acid drug is CpG oligonucleotide modified with cyanine 5 (Cy5-CpG). Among them, the molar ratio of SM102:DSPC:cholesterol:DMG-PEG2000 is 50:10:38.5:1.5, and the nitrogen-phosphorus ratio of SM-102 to the nucleic acid drug is 5.67.

[0020] In an embodiment of the present invention, the negatively charged molecule is selected from one or more of oligodeoxynucleotides and their derivatives.

[0021] In an embodiment of the present invention, the oligodeoxynucleotide includes any short-chain deoxyribonucleic acid DNA with 25 or fewer deoxynucleotides, and can also be represented by ODN. For example, the oligodeoxynucleotide may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 deoxynucleotides.

[0022] In a specific embodiment of the present invention, the oligodeoxynucleotide derivative may be a substance containing any modification known in the art for increasing nuclease reaction activity, improving in vivo stability, enhancing affinity with complementary strands, or enhancing lipid membrane permeability, etc. As an example of the oligodeoxynucleotide derivative, oligodeoxynucleotides with group modifications can be cited, such as amino-modified oligodeoxynucleotides, where an amino group can be modified on the oligodeoxynucleotide, and the amino group can be linked to other ligands activated by NHS ester groups. The amino group and the oligonucleotide can be separated by a C6, C7, or C12 spacer arm to reduce steric hindrance between the amino group and the oligonucleotide. In an embodiment of the present invention, the other ligand is PEG, and the PEG can be PEG 12000, PEG 10000, PEG 8000, PEG 4000, PEG2000, or PEG 1000. Or it can be a thiol-modified oligodeoxynucleotide, where the phosphodiester bond can be replaced by a phosphorothioate bond, a dithiophosphonate bond, a phosphonate alkyl ester bond, an aminophosphonate bond, etc.; or it can be a cholesterol-modified oligodeoxynucleotide, which can be added to the 3' or 5' end of the oligodeoxynucleotide to better insert the oligonucleotide into the lipid membrane structure; or it can be an alkynyl-ring-modified oligodeoxynucleotide, which can be added to the 5' end, so that various azide-modified tags can be added to the alkynyl-ring-modified oligodeoxynucleotide; or it can be a thiol-modified oligodeoxynucleotide, which can be added to the 3' or 5' end of the oligodeoxynucleotide, and the thiol group can be linked to ligands activated by iodoacetamide or maleimide; or it can be a lipid-modified oligodeoxynucleotide, which is added to the 3' or 5' end of the oligodeoxynucleotide, and the lipid part contains an alkyl group, an alkenyl group, an aryl group, or an aralkyl group. The alkenyl group, alkyl group, aryl group, or aralkyl group may contain about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 carbon atoms or more.

[0023] In a specific embodiment of the present invention, the oligodeoxynucleotide derivative is selected from PEG-ODN-cholesterol, amino-ODN, ODN-cholesterol, C14-18 lipid-ODN, thiol-ODN, and / or alkynyl-ODN, etc.

[0024] Among them, the structure of PEG-ODN-cholesterol is shown in formula (I), the structure of amino-ODN is shown in formula (II), and the structure of ODN-cholesterol is shown in formula (III):

[0025]

[0026]

[0027] In one embodiment of the present invention, the preparation method of PEG-ODN-cholesterol comprises: 1) Using a solid-phase carrier modified with amino groups as the reaction starting site, synthesizing amino-ODN by the solid-phase phosphoramidite triester method; 2) Reacting amino-ODN with a cholesterol-modified phosphoramidite monomer to synthesize amino-ODN-cholesterol; 3) Reacting the amino-ODN-cholesterol derivative with an activated polyethylene glycol to generate PEG-ODN-cholesterol;

[0028] Preferably, the ODN in the PEG-ODN-cholesterol consists of 10 thymidines.

[0029] In one embodiment of the present invention, the preparation method of the amino-ODN comprises: Using a solid-phase carrier modified with amino groups as the reaction starting site, synthesizing amino-ODN by the solid-phase phosphoramidite triester method.

[0030] Preferably, the amino-ODN consists of 5'-TCCATGACGTTCCTGACGTT-amino-3'.

[0031] In one embodiment of the present invention, the preparation method of the ODN-cholesterol comprises: 1) Using a solid-phase carrier as the reaction starting site, synthesizing ODN by the solid-phase phosphoramidite triester method; 2) Reacting ODN with a cholesterol-modified phosphoramidite monomer to synthesize ODN-cholesterol;

[0032] Preferably, the ODN in the ODN-cholesterol consists of 5'-TCCATGACGTTCCTGACGTT-3'.

[0033] In one embodiment of the present invention, the surface modification method includes, but is not limited to, modifying negatively charged molecules on the surface of LNP by the directed introduction method, click ligation method or surface insertion method.

[0034] In one embodiment of the present invention, the directed introduction method includes adding a negatively charged molecule to the lipid component for mixing, and assembling to form lipid nanoparticles with negatively charged molecules modified on the surface. For example, the negatively charged molecule is PEG-ODN-cholesterol.

[0035] In one embodiment of the present invention, the click ligation method includes connecting a negatively charged molecule and lipid nanoparticles through a click reaction.

[0036] Preferably, the negatively charged molecule reacts with the polyethylene glycolylated lipid in the lipid nanoparticles;

[0037] Preferably, the polyethylene glycolylated lipid is DSPE-PEG2000-NHS; the negatively charged molecule is amino-ODN.

[0038] In one embodiment of the present invention, the surface insertion method includes connecting negatively charged molecules and lipid nanoparticles through co-incubation.

[0039] Preferably, the negatively charged molecule is ODN-cholesterol.

[0040] According to an embodiment of the present invention, the material of the mineralized shell is an inorganic mineral, preferably an inorganic mineral acceptable to organisms, such as calcium phosphate, calcium carbonate, and / or cadmium sulfide, etc.

[0041] In a second aspect of the present invention, there is provided a method for preparing a composite lipid nanoparticle, comprising the following steps: reacting a lipid nanoparticle surface-modified with a negatively charged molecule with an inorganic ion to form a composite lipid nanoparticle having a mineralized shell.

[0042] Preferably, the surface modification, negatively charged molecule, and lipid nanoparticle all have the definitions as shown above.

[0043] According to an embodiment of the present invention, the inorganic ions include but are not limited to calcium ions (Ca 2+ ) and phosphate ions (PO4 3- ), cadmium ions (Cd 2+ ), and carbonate ions (CO3 2- ).

[0044] For example, a lipid nanoparticle surface-modified with a negatively charged molecule is placed in a mineralization solution and allowed to stand to obtain the composite lipid nanoparticle;

[0045] The mineralization solution contains the above inorganic ions.

[0046] In one embodiment of the present invention, the mineralization solution includes a phosphate buffer solution and a calcium ion solution.

[0047] For example, the mineralization temperature is room temperature, and the mineralization time is not less than 6 h, such as 12 h.

[0048] In a third aspect of the present invention, there is provided a composition, comprising the above composite lipid nanoparticle, an active ingredient;

[0049] and optionally a pharmaceutically acceptable excipient added or not added.

[0050] According to an embodiment of the present invention, the active ingredient is a nucleic acid drug, and preferably the nucleic acid drug is encapsulated within the composite lipid nanoparticle.

[0051] For example, the nucleic acid drug is selected from one or more of a therapeutic agent, a prophylactic agent, or a diagnostic agent.

[0052] In a fourth aspect of the present invention, there is provided the use of the above composite lipid nanoparticle and the above composition in the preparation of a drug or a biological product.

[0053] According to an embodiment of the present invention, the administration routes of the composition, drug or biologic can be intravenous administration, subcutaneous administration, intramuscular administration, inhalation administration, intranasal administration, etc.

[0054] The present invention also provides a drug or biologic prepared from the above-mentioned composite lipid nanoparticles or composition.

[0055] In some embodiments, the biologic is a vaccine, preferably an mRNA vaccine.

[0056] In some embodiments, the biologic can be an influenza vaccine, an AIDS (HIV) vaccine, a viral pneumonia vaccine, a tuberculosis vaccine, a respiratory syncytial virus (RSV) vaccine, an enterovirus (such as EV71) vaccine, a lung cancer vaccine, etc.

[0057] According to an embodiment of the present invention, the drug or biologic is used for preventing and / or treating infectious diseases transmitted through mucous membranes, mucosa-related tumors, respiratory diseases, etc. For example, the infectious diseases are selected from influenza, novel coronavirus infection, pulmonary tuberculosis, bronchitis caused by RSV infection, AIDS virus infection, hand-foot-and-mouth disease caused by enterovirus infection, etc.; for example, the mucosa-related tumors can be oral cancer, lung cancer, etc.; for example, the respiratory diseases can be pulmonary fibrosis, bronchitis, asthma, emphysema, bronchiectasis, byssinosis, hypersensitivity pneumonitis, pleurisy, etc.

[0058] The present invention has the following beneficial effects:

[0059] 1) Based on the four-component LNP used in clinical practice, the present invention, by introducing PEG-ODN-cholesterol molecules directionally during the formation of LNP, and the method of click-connecting amino-ODN molecules or inserting ODN-cholesterol molecules on the surface after the formation of LNP. It can not only efficiently modify negatively charged molecules on the surface of LNP, effectively avoid the encapsulation of negatively charged molecules into the interior of LNP, promote the mineralization of lipid nanoparticles, and improve the stability of LNP (for example, good thermal stability, LNP can be stored at room temperature, and further can be stored at room temperature for a long time); but also improve the cell entry efficiency and enhance the delivery ability of LNP to nucleic acid drugs; at the same time, it can be used as a therapeutic drug to improve the efficacy of LNP, with multiple functions.

[0060] 2) The present invention provides a method for inducing a biomineralization reaction on the surface of LNP to form a mineralized shell. The prepared mineralized shell not only does not affect the drug delivery function of LNP, but also can protect the original structure of LNP.

[0061] Term definitions and explanations

[0062] The term "lipid" refers to a group of organic compounds including, but not limited to, esters of fatty acids, and is characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three classes: (1) "simple lipids", which include fats and oils as well as waxes; (2) "complex lipids", which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0063] The term "LNP" refers to a lipid-nucleic acid particle or nucleic acid-lipid particle (e.g., a stable nucleic acid-lipid particle). An LNP represents a particle made of lipids (e.g., cationic lipids, non-cationic lipids, and conjugated lipids that prevent particle aggregation) and a nucleic acid, wherein the nucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or plasmid, including a plasmid that transcribes interfering RNA or mRNA therefrom) is encapsulated within the lipids. In one embodiment, the nucleic acid is at least 50% encapsulated within the lipids; in one embodiment, the nucleic acid is at least 75% encapsulated within the lipids; in one embodiment, the nucleic acid is at least 90% encapsulated within the lipids; and in one embodiment, the nucleic acid is fully encapsulated within the lipids. LNPs typically contain cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates). LNPs can exhibit an extended circulation lifespan after intravenous (i.v.) injection, can accumulate at distal sites (e.g., sites physically separated from the administration site), and can mediate the expression of the transfected gene or the silencing of target gene expression at these distal sites, playing an extremely important role.

[0064] "Lipid nanoparticle encapsulation" can refer to lipid particles that provide a nucleic acid drug, such as a nucleic acid drug (e.g., interfering RNA or mRNA), in the case of full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated within the lipid nanoparticles (e.g., to form an LNP or other nucleic acid-lipid particle).

[0065] The term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and includes DNA and RNA. DNA can be in the following forms: for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can be in the following forms: siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, viral RNA (vRNA), self-amplifying RNA, and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have binding properties similar to reference nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoroamidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNA).

[0066] The term "surface modification" refers to using physical or chemical methods to change the structure and state of the surface of nanolipid particles to achieve control over the surface of lipid nanoparticles. Currently, there are two main methods for surface modification of LNP. The first method is to add "anchoring" lipids to the LNP formulation, which can be conjugated with functional ligands after particle preparation. Commonly used anchoring lipids include phosphatidylethanolamine, PEG-maleimide, and phosphatidylinositol, which can be chemically coupled with functional ligands. The second method involves preparing the LNP formulation and then inserting hydrophobic antibody derivatives (such as lipid-ligand conjugates) into preformed LNP by optimizing co-incubation. This method does not require reactive anchoring lipids in the LNP formulation and can better control antibody-lipid conjugation.

[0067] The term "biomineralization" refers to the process of converting ions in solution into solid-phase minerals under certain physico-chemical conditions under the control or influence of bioorganic substances. Description of the Drawings

[0068] Figure 1 Hydration particle size diagram of clinical lipid nanoparticles and lipid nanoparticles modified with three negatively charged molecules;

[0069] Figure 2 Surface potential diagram of clinical lipid nanoparticles and lipid nanoparticles modified with three negatively charged molecules;

[0070] Figure 3 Fluorescence signal diagram of FAM-ODN-cholesterol modified lipid nanoparticles;

[0071] Figure 4Morphology diagrams of lipid nanoparticles modified with three negatively charged molecules after mineralization;

[0072] Figure 5 Cell uptake efficiency diagrams of Cy5-CpG in lipid nanoparticles modified with three negatively charged molecules after mineralization;

[0073] Figure 6 Particle sizes of mineralized lipid nanoparticles before and after vortexing for lipid nanoparticles modified with ODN-cholesterol molecules after mineralization. Detailed implementation manners

[0074] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate and explain the present invention exemplarily, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0075] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods.

[0076] Preparation Examples 1-3

[0077] 1. Preparation of PEG-ODN-cholesterol molecules

[0078] By the solid-phase phosphoramidite triester method, using the amino-modified controlled pore glass beads as the starting reaction site, nucleoside phosphoramidite monomers are successively connected to form an ODN composed of 18 thymidines. In the last step of solid-phase synthesis, a cholesterol-modified phosphoramidite monomer is added, so that cholesterol is modified at the 5' end of the ODN, and thus an amino-ODN-cholesterol derivative is obtained. After separation by high-performance liquid chromatography, the purified amino-ODN-cholesterol derivative reacts with N-hydroxysuccinimide-modified polyethylene glycol 2000 (NHS-PEG2000) at a molar ratio of 1:1 in 100 mM sodium bicarbonate buffer (pH 8.0). After separation by agarose gel electrophoresis, the purified target molecule PEG-ODN-cholesterol is obtained. The molecular structure of PEG-ODN-cholesterol is shown in Formula I.

[0079]

[0080] 2. Preparation of amino-ODN molecules

[0081] Using the solid-phase phosphoramidite triester method, with the amino-modified controlled-pore glass beads as the starting reaction site, the nucleoside phosphoramidite monomers are sequentially linked to form an amino-ODN composed of 5'-TCCATGACGTTCCTGACGTT-amino-3' (adenosine (A), thymine (T), cytosine (C), and guanine (G)), and it is separated and purified by high-performance liquid chromatography.

[0082]

[0083] 3. Preparation of ODN-cholesterol molecule

[0084] Using the solid-phase phosphoramidite triester method, with the controlled-pore glass beads without special modification as the starting reaction site, the nucleoside phosphoramidite monomers are sequentially linked to form an ODN composed of 5'-TCCATGACGTTCCTGACGTT-3'. In the last step of solid-phase synthesis, the cholesterol-modified phosphoramidite monomer is added, so that cholesterol is modified at the 5'-end of the ODN, and thus ODN-cholesterol is obtained, and it is separated and purified by high-performance liquid chromatography.

[0085]

[0086] Preparation of four-component lipid nanoparticles in Comparative Example 1

[0087] A comparative example is set up: referring to the preparation method of the Moderna COVID-19 mRNA-1273 formulation, which specifically includes: 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102), distearoyl phosphatidylcholine (DSPC), cholesterol, dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000), and nucleic acid drugs. Among them, the molar ratio of SM102:DSPC:cholesterol:DMG-PEG2000 is 50:10:38.5:1.5, and the nitrogen-phosphorus ratio of SM-102 to the nucleic acid drug is 5.67. The clinical lipid nanoparticles of this comparative example are prepared by the microfluidic method. Among them, the nucleic acid drug is selected as the CpG oligonucleotide modified with cyanine 5 (Cy5-CpG), which is an agonist of Toll-like receptor 9.

[0088] Preparation of lipid nanoparticles modified with negatively charged molecules in Examples 1-3

[0089] Example 1. Preparation of lipid nanoparticles modified with PEG-ODN-cholesterol molecule

[0090] The method of directional introduction is used to modify the negatively charged molecule, that is, 1.5% of PEG-ODN-cholesterol is additionally added to the lipid components of the comparative example based on the total lipid molar amount, and the remaining conditions are the same as those of the comparative example, and it is also prepared by microfluidics.

[0091] The specific preparation method includes 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102), distearoylphosphatidylcholine (DSPC), cholesterol, dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000), PEG-ODN-cholesterol, and a nucleic acid drug. Among them, the molar ratio of SM102:DSPC:cholesterol:DMG-PEG2000:PEG-ODN-cholesterol is 50:10:38.5:1.5:1.5, and the nitrogen-phosphorus ratio of SM-102 to the nucleic acid drug is 5.67. Clinical lipid nanoparticles are prepared by the microfluidic method. Among them, the nucleic acid drug is selected as CpG oligonucleotide modified with cyanine 5 (Cy5-CpG), which is an agonist of Toll-like receptor 9.

[0092] Example 2. Preparation of lipid nanoparticles modified with amino-ODN molecules

[0093] The negatively charged molecule is modified by the click-connection method. Replace the DMG-PEG2000 component in the preparation method of the comparative example with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-hydroxysuccinimide-polyethylene glycol 2000 (DSPE-PEG2000-NHS) with the same molar amount, and keep the other conditions the same as those of the comparative example, and it is also prepared by microfluidics. After the formation of the lipid nanoparticles, add amino-ODN with the same molar amount as DSPE-PEG2000-NHS and oscillate in phosphate buffer (pH 7.4) for 12 h to connect the amino-ODN to the surface of the lipid nanoparticles, and remove the unreacted amino-ODN by ultrafiltration through a 30000 Da ultrafiltration tube.

[0094] Example 3. Preparation of lipid nanoparticles modified with ODN-cholesterol molecules

[0095] The negatively charged molecule is modified by the surface insertion method. After mixing the lipid nanoparticles in the preparation method of the comparative example with ODN-cholesterol accounting for 1.5% of the total lipid molar amount, oscillate at room temperature in a high-concentration salt solution for 12 h to insert the ODN-cholesterol into the surface of the lipid nanoparticles, and remove the uninserted ODN-cholesterol by ultrafiltration through a 30000 Da ultrafiltration tube.

[0096] The specific preparation method includes: 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102), distearoylphosphatidylcholine (DSPC), cholesterol, dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000), and a nucleic acid drug. Among them, the molar ratio of SM102:DSPC:cholesterol:DMG-PEG2000 is 50:10:38.5:1.5, and the nitrogen-phosphorus ratio of SM-102 to the nucleic acid drug is 5.67. Lipid nanoparticles are prepared by the microfluidic method. Among them, the nucleic acid drug is selected as CpG oligonucleotide modified with cyanine 5 (Cy5-CpG), which is an agonist of Toll-like receptor 9. After mixing the lipid nanoparticles and ODN-cholesterol (the molar ratio of SM102:DSPC:cholesterol:DMG-PEG2000:cholesterol is 50:10:38.5:1.5:1.5), they are oscillated at room temperature for 12 h in a high-concentration salt solution to insert ODN-cholesterol onto the surface of the lipid nanoparticles, and the uninserted ODN-cholesterol is removed by ultrafiltration through a 30000 Da ultrafiltration tube.

[0097] Test Example 1 Hydrodynamic diameter and surface potential of clinical lipid nanoparticles and negatively charged modified lipid nanoparticles

[0098] 1. Respectively take 60 μL of the clinical lipid nanoparticles in Comparative Example 1 and the three negatively charged modified lipid nanoparticles in Examples 1-3 and add them to a micro-sample cell. The hydrodynamic diameters of the four different lipid nanoparticles in phosphate buffer were measured by dynamic light scattering method. The results are shown in Figure 1 .

[0099] From Figure 1 it can be seen that despite the different modification methods, the finally obtained negatively charged modified lipid nanoparticles still maintain a stable and uniform nanostructure.

[0100] 2. Respectively take 600 μL of the clinical lipid nanoparticles in Comparative Example 1 containing 10 ng / μL Cy5-CpG and the three negatively charged modified lipid nanoparticles in Examples 1-3 and add them to an electric potential cell. The surface potentials of the four lipid nanoparticles in 0.1× phosphate buffer were measured using a Zetasizer Nano ZSP instrument. The results are shown in Figure 2 .

[0101] From Figure 2 it can be seen that compared with the clinical lipid nanoparticles, the surface potential of the negatively charged modified lipid nanoparticles is significantly reduced.

[0102] Test Example 2 Gel electrophoresis characterization of the stability of negatively charged molecules modified onto lipid nanoparticles

[0103] The 5-carboxyfluorescein molecule was used to modify the lipid nanoparticles modified with ODN-cholesterol in Example 3. The specific method includes:

[0104] After modifying the 3'-end of the ODN-cholesterol molecule with a 5-carboxyfluorescein (5-FAM) molecule, FAM-ODN-cholesterol was obtained. Then, FAM-ODN-cholesterol was inserted onto the surface of the clinical lipid nanoparticles encapsulating Cy5-CpG by the surface insertion method. Take 10 μL of the negatively charged modified lipid nanoparticles thus obtained and add them to the loading well of a 3% agarose gel. After electrophoresis for 30 minutes at a voltage of 100 V, the distribution of FAM and Cy5 fluorescence signals was characterized by a fluorescence imager. The results are shown in Figure 3 .

[0105] From Figure 3 it can be seen that both the FAM signal and the Cy5 signal remained in the loading well and did not move with electrophoresis, indicating that the inserted FAM-ODN-cholesterol could stably bind to the surface of the lipid nanoparticles.

[0106] Testing Example 3 Mineralization of negatively charged modified lipid nanoparticles and their morphology and elemental composition

[0107] The three kinds of negatively charged modified lipid nanoparticles in Examples 1-3 were respectively resuspended in 0.1× phosphate buffer (containing 0.1 mM phosphate), and an aqueous calcium chloride solution equimolar to the phosphate was added. After standing at room temperature for 12 h, lipid nanoparticles protected by a mineralized shell were formed. The morphology of the mineralized lipid nanoparticles was observed by transmission electron microscopy, and the elemental composition of the particles was analyzed by combining with an X-ray energy spectrometer. The results are shown in Figure 4 .

[0108] From Figure 4 it can be known that the mineralized lipid nanoparticles formed a burr-like shell. In addition to the N, O, and P originally contained in the lipid nanoparticles, a large amount of Ca element was also present, indicating that the formed outer burr-like shell was mainly an inorganic structure of calcium.

[0109] Testing Example 4 Cellular uptake efficiency of negatively charged modified lipid nanoparticles and mineralized negatively charged modified lipid nanoparticles

[0110] 100 mmol of the mineralized lipid nanoparticles modified with ODN-cholesterol in Example 3, the lipid nanoparticles modified with ODN-cholesterol in Example 3, the clinical lipid nanoparticles in Comparative Example 1, and Cy5-CpG were respectively added to a 96-well plate. After co-incubating with 50,000 dendritic cells for 3 h, the Cy5-CpG content taken up by the cells was analyzed by flow cytometry. The results are shown in Figure 5 .

[0111] From Figure 5It can be seen that compared with clinical lipid nanoparticles, the efficiency of negatively charged modified lipid nanoparticles entering cells has been significantly improved, and the cell efficiency of mineralized lipid nanoparticles is still significantly higher than that of clinical lipid nanoparticles, indicating that the mineralized lipid nanoparticles still maintain the efficient nucleic acid delivery function of negatively charged lipid nanoparticles.

[0112] Test Example 5 Stability of Mineralized Negatively Charged Modified Lipid Nanoparticles

[0113] To test the influence of external force on the mineralized lipid nanoparticles, 500 μL of the mineralized lipid nanoparticles modified with ODN-cholesterol molecules in Example 3 were vortexed at 3000 rpm for 60 seconds, and then the particle sizes of the mineralized lipid nanoparticles before and after vortexing were detected by a dynamic light scattering instrument. The results are shown in Figure 6 .

[0114] As can be seen from Figure 6 it, the particle size of the mineralized lipid nanoparticles basically does not change after vortexing, indicating that the mineralized lipid nanoparticles have good stability and are not easily agglomerated and damaged by external forces.

[0115] At the same time, 500 μL of the mineralized lipid nanoparticles modified with ODN-cholesterol molecules in Example 3 were heated at 55 °C for 6 hours, and then the particle sizes of the mineralized lipid nanoparticles before and after heating were detected by a dynamic light scattering instrument. The results show that the particle size of the heated lipid nanoparticles basically does not change, indicating that the mineralized lipid nanoparticles have good thermal stability.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A composite lipid nanoparticle, characterized in that, The composite lipid nanoparticles are composed of lipid nanoparticles modified with negatively charged molecules on the surface and a mineralized shell deposited on the surface of the lipid nanoparticles.

2. The composite lipid nanoparticle according to claim 1, wherein, The lipid nanoparticles include a lipid component, and the lipid component includes an ionizable lipid, a helper phospholipid, cholesterol and its derivatives, and a polyethylene glycolylated lipid; Preferably, the ionizable lipid accounts for 20 mol% to 80 mol% of the lipid component in the lipid nanoparticles; Preferably, the ionizable lipid includes one or more of 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (nonadec-9-yl) ester (SM-102), [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 4-(N,N-dimethylamino)butyric acid (dilinoleoyl) methyl ester (DLin-MC3-DMA), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12), 9-(4-(dimethylamino)butanoyloxy)heptadecanedioic acid bis((Z)-non-2-en-1-yl) ester (L319), N2,2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoic acid (nonadec-9-yl) ester (Lipid5), 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl)azanediyl]bis(dodecan-2-ol) (C12-200), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium bromide (DDAB), tetra(8-methylnonyl) 3,3',3”,3”'-{[(methylazanediyl)bis(propane-3,1-diyl)]bis(azanetriyl)}tetrapropionate (306Oi10); Preferably, the helper phospholipid accounts for 2 mol% to 30 mol% of the lipid component in the lipid nanoparticles; Preferably, the helper phospholipid includes, but is not limited to, one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); Preferably, the cholesterol and its derivatives account for 10 mol% to 50 mol% of the lipid components in the lipid nanoparticles; Preferably, the cholesterol and its derivatives include, but are not limited to, one or more of β-sitosterol, cholestanol, cholestanone, cholesterol, cholestenone, 7β-hydroxy cholesterol, and 7α-hydroxy cholesterol; Preferably, the polyethylene glycolated lipid accounts for 0.3 mol% - 30 mol% of the lipid components in the lipid nanoparticles; Preferably, the polyethylene glycolated lipid includes, but is not limited to, one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol, 1,2-dipalmitoyl-rac-glycerol-3-methoxypolyethylene glycol, and 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine-methoxypolyethylene glycol; Preferably, the lipid nanoparticles further include a nucleic acid drug, the lipid nanoparticles encapsulate the nucleic acid drug, and the nucleic acid drug includes antisense nucleic acid, small interfering RNA, microRNA, small activating RNA, messenger RNA, and aptamer; Preferably, the N / P ratio of the lipid nanoparticles is 4 - 16:1; More preferably, the ionizable lipid is 1-octyl nonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102), the co-lipid is distearoyl phosphatidylcholine (DSPC), cholesterol, the polyethylene glycolated lipid is dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000), and the nucleic acid drug is CpG oligonucleotide modified with cyanidin 5, wherein the molar ratio of SM102:DSPC:cholesterol:DMG-PEG2000 is 50:10:38.5:1.5, and the nitrogen-phosphorus ratio of SM-102 to the nucleic acid drug is 5.

67.

3. A composite lipid nanoparticle according to claim 1, wherein, The negatively charged molecule is selected from one or more than two of oligodeoxynucleotides and their derivatives; Preferably, the oligodeoxynucleotide includes any short-chain deoxyribonucleic acid DNA with 25 or fewer deoxynucleotides; Preferably, the oligodeoxynucleotide derivative includes an oligodeoxynucleotide modified with a group, and the group modification includes amino modification, thiol modification, cholesterol modification, alkynyl modification, mercapto modification, and lipid modification; More preferably, the oligodeoxynucleotide derivative includes PEG-ODN-cholesterol, amino-ODN, and / or ODN-cholesterol, C14 - C18 lipid-ODN, mercapto-ODN, and / or alkynyl-ODN; 4. The composite lipid nanoparticle according to claim 3, characterized in that, The oligodeoxynucleotide derivative includes PEG-ODN-cholesterol, amino-ODN, and ODN-cholesterol, wherein the structure of PEG-ODN-cholesterol is shown in formula (I), the structure of amino-ODN is shown in formula (II), and the structure of ODN-cholesterol is shown in formula (III): Preferably, the method for preparing PEG-ODN-cholesterol comprises: 1) Using an amino-modified solid support as the reaction starting site, synthesizing amino-ODN by solid-phase phosphoramidite triester method; 2) Reacting amino-ODN with a cholesterol-modified phosphoramidite monomer to synthesize amino-ODN-cholesterol; 3) Reacting the amino-ODN-cholesterol derivative with activated polyethylene glycol to generate PEG-ODN-cholesterol; Preferably, the ODN in the PEG-ODN-cholesterol consists of 10 thymidines; Preferably, the method for preparing the amino-ODN comprises: Using an amino-modified solid support as the reaction starting site, synthesizing amino-ODN by solid-phase phosphoramidite triester method; Preferably, the amino-ODN consists of 5'-TCCATGACGTTCCTGACGTT-amino-3'; Preferably, the method for preparing the ODN-cholesterol comprises: 1) Using a solid support as the reaction starting site, synthesizing ODN by solid-phase phosphoramidite triester method; 2) Reacting ODN with a cholesterol-modified phosphoramidite monomer to synthesize ODN-cholesterol; Preferably, the ODN in the ODN-cholesterol consists of 5'-TCCATGACGTTCCTGACGTT-3'; 5. A composite lipid nanoparticle according to claim 1, wherein The surface modification method includes, but is not limited to, modifying negatively charged molecules on the surface of LNP by directed introduction method, click ligation method or surface insertion method; Preferably, the directed introduction method includes adding negatively charged molecules to the lipid component for mixing, and assembling to form lipid nanoparticles with negatively charged molecules modified on the surface; Preferably, the click ligation method includes connecting negatively charged molecules and lipid nanoparticles by click reaction; More preferably, the negatively charged molecules react with the polyethylene glycolated lipid in the lipid nanoparticles; Further preferably, the polyethylene glycolated lipid is DSPE-PEG2000-NHS; The negatively charged molecule is amino-ODN; Preferably, the surface insertion method includes connecting negatively charged molecules and lipid nanoparticles by co-incubation; More preferably, the negatively charged molecule is ODN-cholesterol.

6. A composite lipid nanoparticle according to claim 1, wherein, The material of the mineralized shell is an inorganic mineral, preferably an inorganic mineral acceptable to organisms, more preferably calcium phosphate, calcium carbonate, cadmium sulfide.

7. A method for preparing the composite lipid nanoparticles according to any one of claims 1-6, comprising the following steps: Reacting the lipid nanoparticles with negatively charged molecules modified on the surface with inorganic ions to form composite lipid nanoparticles with a mineralized shell; Preferably, the inorganic ions include calcium ions, phosphate ions, cadmium ions, carbonate ions.

8. A composition, characterized in that, Comprising the composite lipid nanoparticles according to any one of claims 1-6, an active ingredient; and optionally added or not added pharmaceutically acceptable excipients.

9. Use of the composite lipid nanoparticles according to any one of claims 1-6 and the composition according to claim 8 in the preparation of a drug or a biological product.

10. A drug or biological product, characterized in that, Prepared from the composite lipid nanoparticles according to any one of claims 1-6 or the composition according to claim 8; Preferably, the biological product is a vaccine, more preferably an mRNA vaccine; Further preferably, the biological product is an influenza vaccine, an AIDS vaccine, a viral pneumonia vaccine, a tuberculosis vaccine, a respiratory syncytial virus vaccine, an enterovirus vaccine, or a lung cancer vaccine.

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