CN117257761A - Nucleic acid-entrapped antioxidant lipid nanoparticle, and preparation method and application thereof - Google Patents

Nucleic acid-entrapped antioxidant lipid nanoparticle, and preparation method and application thereof Download PDF

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CN117257761A
CN117257761A CN202311211551.8A CN202311211551A CN117257761A CN 117257761 A CN117257761 A CN 117257761A CN 202311211551 A CN202311211551 A CN 202311211551A CN 117257761 A CN117257761 A CN 117257761A
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lipid
parts
peg
nucleic acid
polyethylene glycol
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陈小元
喻国灿
于馨洋
戚少龙
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Shanghai Theranostics Biotechnology Co ltd
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Shanghai Theranostics Biotechnology Co ltd
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Abstract

The invention provides a raw material composition for preparing antioxidant lipid nanoparticles, which comprises the following components in parts by weight: 10-70 parts of ionized lipid, 1-30 parts of neutral lipid, 1-20 parts of polyethylene glycol lipid, 5-60 parts of steroid lipid and 1-50 parts of antioxidant. The invention also provides a nucleic acid drug delivery system, which is spherical particles with the particle diameter of 50-100nm, composed of LNP-entrapped nucleic acid molecules; the LNP comprises, by weight, 10-70 parts of ionizable lipid, 10-30 parts of neutral lipid, 1-20 parts of polyethylene glycol lipid, 5-60 parts of steroid lipid and 1-50 parts of antioxidant. The invention also provides a method for preparing the nucleic acid drug delivery system, and application of the raw material composition in preparing vaccines or genetically modified immune cells, or application in inducing reprogramming and redifferentiation of multifunctional stem cells (iPSCs) of various primary cells. The nucleic acid drug delivery system has sufficient stability, safety and higher loading efficiency, and can obviously improve the presenting capability of cell antigens and the immune response of organisms.

Description

Nucleic acid-entrapped antioxidant lipid nanoparticle, and preparation method and application thereof
Technical Field
The invention relates to the fields of biological medicine, nanotechnology, supermolecule assembly and nucleic acid delivery, in particular to a nucleic acid-entrapped lipid nanoparticle with an antioxidant effect, a preparation method thereof and application thereof in nucleic acid delivery.
Background
Lipid nanoparticles (Lipid nanoparticles, LNP) are widely used for vaccine development because of their relative safety, high efficiency and ease of phagocytosis by antigen presenting cells. Currently, LNP delivery technology is adopted by three major mRNA vaccine megahead enterprises, moderna, cureVac and BioNTech. By the end of 2021, over 20 million people worldwide have vaccinated with the covd-19 mRNA vaccine delivered by LNP, with a total sales of over 600 million dollars. However, with the wide and deep application, the defects of the method are gradually revealed: (1) existing LNP delivery systems can lead to severe liver accumulation, which in turn can lead to liver injury or the occurrence of immune hepatitis. There are continuous reports showing that the novel crown mRNA vaccines of Pfizer/BioNTech and Moderna induce liver injury and immune hepatitis, and that liver dysfunction also occurs after part of the population is vaccinated with the mRNA vaccine. (2) Lnp@mrna vaccine is less enriched in Dendritic Cells (DCs) after intramuscular injection, and more enriched in muscle cells, which may also cause allergic and inflammatory reactions while impairing the vaccine effect. In addition, the generation of inflammatory responses in turn may lead to the recruitment of monocyte clusters such as macrophages, centromeres, leukocytes, etc., thereby exacerbating non-targeted phagocytosis of LNP, further impairing the immune efficacy of the vaccine. Thus, reducing the inflammatory response at the injection site and systemic non-target sites is critical to enhance the immune activation effect of LNP.
Studies have shown that oxidative stress is associated with a number of acute and chronic inflammatory diseases. Reactive oxygen species (reactive oxygen species, ROS) are single electron reduction products of a class of oxygen in the body, including superoxide anions (O) 2- ) Hydrogen peroxide (H) 2 O 2 ) Hydroxyl radicals (OH. Cndot.), and the like. ROS have a very wide range of effects on the body as a byproduct of normal metabolism, resulting in various endogenous injuries such as DNA damage, protein damage, etc. Excessive accumulation of ROS leads to cell and tissue damage, thereby triggering an inflammatory response.At the same time, the inflammatory response also produces large amounts of ROS, further exacerbating local tissue damage and even causing chronic inflammation. Antioxidants can slow or prevent the damage of redox or ROS to cells and active substances in the organism. Antioxidants are typically reducing agents such as mercaptans, ascorbic acid, polyphenols, and the like. These reducing agents are capable of nonspecific chain scission and oxidation resistance, and prevent free radical reaction, thereby protecting polyunsaturated fatty acids in cell membrane phospholipids and plasma lipoproteins from attack by oxygen radicals. Given the various inflammatory side effects of LNP during application and the powerful anti-redox properties of antioxidants in organisms, integration of the functions of LNP and antioxidants will result in a "1+1" combination >2'.
Disclosure of Invention
To address the deficiencies of existing LNP delivery technologies, the immune activation efficacy of LNP is improved. The primary object of the present invention is to provide a raw material composition for preparing lipid nanoparticles having an antioxidant effect and capable of remarkably improving the antigen presenting ability of cells, and a nucleic acid drug delivery system.
Another object of the present invention is to provide a method for preparing the nucleic acid drug delivery system, so as to obtain nucleic acid-entrapped lipid nanoparticles having good antioxidant efficacy by a simple and easy method.
It is a further object of the present invention to provide the use of said raw material composition for the preparation of a vaccine.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect, the present invention provides a feedstock composition for preparing antioxidant lipid nanoparticles, the components of which comprise an ionizable lipid, a neutral lipid, a polyethylene glycol lipid, a steroid lipid, and an antioxidant.
In the raw material composition, the raw material composition comprises the following components in parts by weight: 10-70 parts of ionizable lipid, 1-30 parts of neutral lipid, 1-20 parts of polyethylene glycol lipid, 5-60 parts of steroid lipid and 1-50 parts of antioxidant.
In the preferred raw material composition, the weight parts of the components are as follows: 30-60 parts of ionizable lipid, 5-15 parts of neutral lipid, 1-15 parts of polyethylene glycol lipid, 10-50 parts of steroid lipid and 2-50 parts of antioxidant.
In the more preferred raw material composition of the invention, the following components are mixed in parts by weight: 40-55 parts of ionizable lipid, 8-10 parts of neutral lipid, 5-15 parts of polyethylene glycol lipid, 10-30 parts of steroid lipid and 5-30 parts of antioxidant.
In the most preferred raw material composition of the invention, the following components are mixed according to parts by weight: 54 parts of ionizable lipid, 10 parts of neutral lipid, 11 parts of polyethylene glycol lipid, 20 parts of steroid lipid and 5 parts of antioxidant.
In the raw material composition of the present invention, the antioxidant may be selected from any one of the following: vitamin A (VA) or a derivative thereof, vitamin C (VC) or a derivative thereof, vitamin E (VE) or a derivative thereof, coenzyme Q10 (UQ) or a derivative thereof, lipoic Acid (LA) or a derivative thereof, or polyphenol (PP) or a derivative thereof, or a mixture of any two or more thereof; preferably vitamin E or a derivative thereof, coenzyme Q10 or a derivative thereof or lipoic acid or a derivative thereof; further preferred is vitamin E or a derivative thereof, or lipoic acid or a derivative thereof; most preferred is vitamin E or a derivative thereof.
The Vitamin A (VA) or its derivative may be specifically selected from: any one or a mixture of two or more of retinol (retinol), retinol aldehyde (retinaldehyde), vitamin a ester (retinester), vitamin a propionate (retinol propionate), trans-retinoic acid (tretinoin), adapalene (adapalene), tazarotene (tazarote), and the like.
The Vitamin C (VC) or its derivatives may be specifically selected from: any one or a mixture of two or more of ascorbyl glucoside, ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, trioxyethyl ascorbate, tetrahexyldecyl ascorbate, tetraisopalmitate ascorbate, trisodium ascorbyl palmitate phosphate, aminopropanol ascorbyl phosphate, diglycerol ascorbate, hexylglycerol ascorbate, and disodium isostearyl ascorbate.
The vitamin E or its derivative may be specifically selected from: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, tocopherol acetate, tocopherol palmitate, D-alpha-vitamin E, DL-alpha-vitamin E, vitamin E polyethylene glycol succinate, vitamin E acetate, D-gamma-vitamin E, vitamin E acetate, vitamin E succinate, alpha-vitamin E nicotinate, or the like.
The lipoic acid or derivative thereof may be selected from lipoic acid polymers or derivatives thereof having various side chain groups (e.g., cyclodextrin, carboxyl, amine, azide, or other functional groups).
The polyphenol or derivative thereof may be selected from: any one or more of tannic acid, dopamine, catechol, gallic acid, catechin, protocatechuic aldehyde, ellagic acid or procyanidine.
In the raw material composition of the present invention, the ionizable lipid may be selected from any of the lipid molecules represented by the following lipid skeletons containing one or more ionizable sites (including primary amine, secondary amine, tertiary amine, etc.): c12-200, ALC-0315, SM102, DODAP, DODMA, DOBAQ, YSK05, dlin-DMA, dlin-KC2-DMA or Dlin-MC3-DMA, or a mixture of any two or more thereof.
In the raw material composition of the present invention, the neutral lipid may be selected from any one or more of the following compositions: 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), or 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
In the raw material composition of the invention, the polyethylene glycol lipid can be selected from any one or more of the following compositions: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerideamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxy propyl-3-amine (PEG-c-DMA).
In the raw material composition of the present invention, the steroid lipid may be selected from any one or a mixture of two or more of the following: oat sterols, beta-sitosterols, campesterols, ergocalcitols, campesterols, cholestanol, cholesterol, fecal sterols, dehydrocholesterol, desmosterols, dihydroergocalcitols, dihydrocholesterol, dihydroergosterols, black-sea sterols, episterols, ergosterols, fucosterols, hexahydrophotopterols, hydroxycholesterols; lanosterol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, or lithocholic acid.
The raw material composition of the present invention may be in various forms obtained by mixing the components in the above-mentioned proportions, and may be, for example, a solid composition or a solution obtained by dissolving the components in a solvent.
In a second aspect, the present invention provides a nucleic acid drug delivery system, which is spherical particles with a particle size of 50-100nm, comprising LNP-entrapped nucleic acid molecules; wherein the LNP is composed of ionizable lipid, neutral lipid, polyethylene glycol lipid, steroid lipid and antioxidant; the weight ratio of LNP to nucleic acid molecule is 5:1-50:1; preferably 10:1 to 30:1; most preferably 25:1.
In the nucleic acid drug delivery system, the LNP comprises, by weight, 10-70 parts of ionizable lipid, 1-30 parts of neutral lipid, 1-20 parts of polyethylene glycol lipid, 5-60 parts of steroid lipid and 1-50 parts of antioxidant.
In a preferred nucleic acid drug delivery system of the present invention, the LNP is composed of, by weight, 30-60 parts of an ionizable lipid, 5-15 parts of a neutral lipid, 1-15 parts of a polyethylene glycol lipid, 10-50 parts of a steroid lipid, and 2-50 parts of an antioxidant.
In a more preferred nucleic acid drug delivery system of the present invention, the LNP is composed of, by weight, 40-55 parts of an ionizable lipid, 8-10 parts of a neutral lipid, 5-15 parts of a polyethylene glycol lipid, 10-30 parts of a steroid lipid, and 5-30 parts of an antioxidant.
In the most preferred nucleic acid drug delivery system of the present invention, the starting material for LNP consists of 54 parts by weight of ionizable lipid, 10 parts by weight of neutral lipid, 11 parts by weight of polyethylene glycol lipid, 20 parts by weight of steroid lipid, and 5 parts by weight of antioxidant.
According to the invention, the antioxidant is added into the LNP raw material in the proportion, so that the LNP nucleic acid-coated nucleic acid drug delivery system has an antioxidation effect, local inflammatory reaction is reduced, and mRNA vaccine expression efficiency is further improved.
In the nucleic acid drug delivery system of the present invention, the antioxidant may be a compound having an anti-redox effect such as Vitamin A (VA) or a derivative thereof, vitamin C (VC) or a derivative thereof, vitamin E (VE) or a derivative thereof, coenzyme Q10 (UQ) or a derivative thereof, lipoic Acid (LA) or a derivative thereof, or polyphenol PP (Polyphenols PP) or a derivative thereof, and may be specifically selected from any one or a combination of two or more of the following molecules:
vitamin a or a derivative thereof: retinol (retinol), retinol aldehyde (retinaldehyde), vitamin a ester (retinyl ester), vitamin a propionate (retinol propionate), trans-retinoic acid (tretinoin), adapalene (adapalene) or tazarote (tazarote), and the like.
Vitamin C or a derivative thereof: ascorbyl glucoside, ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, trioxyethyl ascorbate, tetrahexyldecyl ascorbate, tetraisopalmitate ascorbate, trisodium ascorbyl palmitate phosphate, aminopropanol ascorbyl phosphate, diglycerol ascorbate, hexylglycerol ascorbate, or disodium isostearyl ascorbyl phosphate, and the like.
Vitamin E or a derivative thereof: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, tocopherol acetate, tocopherol palmitate, D-alpha-vitamin E, DL-alpha-vitamin E, vitamin E polyethylene glycol succinate, vitamin E acetate, D-gamma-vitamin E, vitamin E acetate, vitamin E succinate, alpha-vitamin E nicotinate, and the like.
Lipoic acid or a derivative thereof: lipoic acid polymers or derivatives thereof containing different side chain groups (e.g., cyclodextrin, carboxyl, amine, azide, or other functional groups).
Polyphenols or derivatives thereof: tannic acid, dopamine, catechol, gallic acid, catechin, protocatechuic aldehyde, ellagic acid or procyanidin, etc.
In the nucleic acid drug delivery system of the present invention, the ionizable lipid may be selected from the following lipid molecules represented by a lipid backbone containing one or more ionizable sites (including primary amine, secondary amine, tertiary amine, etc.), and the ionizable lipid may be specifically selected from the group consisting of: : any one or more of C12-200, ALC-0315, SM102, DODAP, DODMA, DOBAQ, YSK05, dlin-DMA, dlin-KC2-DMA or Dlin-MC 3-DMA.
In the nucleic acid drug delivery system of the present invention, the polyethylene glycol lipid may be selected from: any one or more than two of 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerideamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).
In the nucleic acid drug delivery system of the present invention, the neutral lipid may be selected from the group consisting of: any one or a combination of more than two of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycero) (DOPG), oleoyl phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
In the nucleic acid drug delivery system of the present invention, the steroid lipid may be selected from: oat sterols, beta-sitosterols, campesterols, ergocalcitols, campesterols, cholestanol, cholesterol, fecal sterols, dehydrocholesterol, desmosterols, dihydroergocalcitols, dihydrocholesterol, dihydroergosterols, black-sea sterols, episterols, ergosterols, fucosterols, hexahydrophotopterols, hydroxycholesterols; lanosterol, photosterol, sitosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid.
In the nucleic acid drug delivery system of the present invention, the nucleic acid molecule may be a variety of exogenous nucleic acid molecules, including DNA nucleic acid molecules and RNA nucleic acid molecules. The DNA nucleic acid molecule may be a plasmid, a single-stranded DNA molecule, or a double-stranded DNA molecule. The RNA nucleic acid molecule may be a protein-encoding linear RNA, a protein-encoding circular RNA, a self-replicating RNA, or various non-encoding RNAs (e.g., microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs or long-chain non-encoding RNAs). The RNA molecule can be provided with various base modifications or cap structures, and the base modifications can be as follows: methylation modifications (such as N6-methyl adenosine (M6A), N1-methyl adenosine (M1A), 5-methyl cytidine (M5C), 3-methyl cytidine (M3C), N7-methyl guanosine (M7G) or 1-methyl guanosine (M1G), 2 '-O-methyl guanosine or N6,2' -O-dimethyl guanosine (M6 Am)), methoxyethoxy modifications (such as 2-methoxyethoxyadenosine, 2-methoxyethoxycytidine, 2-methoxyethoxyguanosine, 2-methoxyethoxyuridine), fluoridation modifications, pseudouracil modifications (ψ), or methyl pseudouracil modifications (M1- ψ), the cap structure may be cap0, cap1 or cap2 cap structures.
In a third aspect, the present invention provides a method of preparing a nucleic acid drug delivery system according to the second aspect of the invention, comprising:
1) Dissolving a mixture of 10-70% ionizable lipid, 1% -30% neutral lipid, 1% -20% polyethylene glycol lipid, 5% -60% steroid lipid and 1% -50% antioxidant, preferably a mixture of 30% -60% ionizable lipid, 5% -15% neutral lipid, 1% -15% polyethylene glycol lipid, 10% -50% steroid lipid and 2% -50% antioxidant, more preferably a mixture of 40% -55% ionizable lipid, 8% -10% neutral lipid, 5% -15% polyethylene glycol lipid, 10% -30% steroid lipid and 5% -30% antioxidant, most preferably a mixture of 54% ionizable lipid, 10% neutral lipid, 11% polyethylene glycol lipid, 20% steroid lipid and 5% antioxidant in an organic solvent to obtain a lipid solution containing a visual oxidant;
2) Dissolving nucleic acid in a buffer solution having a pH of 3 to 9, preferably in a buffer solution having a pH of 4 to 6, more preferably in a buffer solution having a pH of 5, to obtain a nucleic acid solution;
3) Mixing the lipid solution obtained in the step 1) with the nucleic acid solution obtained in the step 2), and controlling the weight ratio of the total lipid containing the antioxidant to the nucleic acid molecules to be 5:1-50:1 after mixing; and then the mixed solution is co-extruded by utilizing a microfluidic technology to prepare spherical particles, namely the LNPs for coating the nucleic acid molecules.
In the preparation method of the present invention, the antioxidant 1) may be a compound having an anti-redox effect, such as Vitamin A (VA) or a derivative thereof, vitamin C (VC) or a derivative thereof, vitamin E (VE) or a derivative thereof, coenzyme Q10 (UQ) or a derivative thereof, lipoic Acid (LA) or a derivative thereof, or polyphenol PP (Polyphenols PP) or a derivative thereof, and may specifically be selected from any one or a combination of two or more of the following molecules:
vitamin a or a derivative thereof: retinol (retinol), retinol aldehyde (retinaldehyde), vitamin a ester (retinyl ester), vitamin a propionate (retinol propionate), trans-retinoic acid (tretinoin), adapalene (adapalene) or tazarote (tazarote), etc.;
vitamin C or a derivative thereof: ascorbyl glucoside, ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, trioxyethyl ascorbate, tetrahexyldecyl ascorbate, tetraisopalmitate ascorbyl palmitate, trisodium ascorbyl palmitate, ascorbyl aminopropanol phosphate, diglyceryl ascorbate, hexylglyceryl ascorbate, disodium isostearyl ascorbate, and the like;
Vitamin E or a derivative thereof: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, tocopherol acetate, tocopherol palmitate, D-alpha-vitamin E, DL-alpha-vitamin E, vitamin E polyethylene glycol succinate, vitamin E acetate, D-gamma-vitamin E, vitamin E acetate, vitamin E succinate, or alpha-vitamin E nicotinate, and the like;
lipoic acid or a derivative thereof: lipoic acid polymers or derivatives thereof containing different side chain groups (e.g., cyclodextrin, carboxyl, amine, azide, or other functional groups);
polyphenols or derivatives thereof: tannic acid, dopamine, catechol, gallic acid, catechin, protocatechuic aldehyde, ellagic acid or procyanidin, etc.;
in the preparation method of the present invention, the ionizable lipid 1) may be selected from the following lipid molecules represented by a lipid skeleton containing one or more ionizable sites (including primary amine, secondary amine, tertiary amine, etc.), and the ionizable lipid may be specifically selected from: any one or more of C12-200, ALC-0315, SM102, DODAP, DODMA, DOBAQ, YSK05, dlin-DMA, dlin-KC2-DMA or Dlin-MC 3-DMA.
In the preparation method of the present invention, the polyethylene glycol lipid of 1) may be selected from: any one or more than two of 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerideamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).
In the preparation method of the present invention, the neutral lipid of 1) may be selected from: any one or a combination of more than two of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycero) (DOPG), oleoyl phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
In the preparation method of the present invention, the steroid lipid of 1) may be selected from: oat sterols, beta-sitosterols, campesterols, ergocalcitols, campesterols, cholestanol, cholesterol, fecal sterols, dehydrocholesterol, desmosterols, dihydroergocalcitols, dihydrocholesterol, dihydroergosterols, black-sea sterols, episterols, ergosterols, fucosterols, hexahydrophotopterols, hydroxycholesterols; lanosterol, photosterol, sitosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid.
In the preparation method of the invention, the organic solvent of 1) can be selected from methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide or N, N-dimethylformamide; preferably ethanol, tetrahydrofuran or acetone; most preferred is ethanol.
In the preparation method of the present invention, the nucleic acid molecule of 2) may be a variety of exogenous nucleic acid molecules including DNA nucleic acid molecules and RNA nucleic acid molecules. The DNA nucleic acid molecule may be a plasmid, a single-stranded DNA molecule, or a double-stranded DNA molecule. The RNA nucleic acid molecule may be a protein-encoding linear RNA, a protein-encoding circular RNA, a self-replicating RNA, or various non-encoding RNAs (e.g., microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs or long-chain non-encoding RNAs). The RNA molecule can be provided with various base modifications or cap structures, and the base modifications can be as follows: methylation modifications (such as N6-methyl adenosine (M6A), N1-methyl adenosine (M1A), 5-methyl cytidine (M5C), 3-methyl cytidine (M3C), N7-methyl guanosine (M7G) or 1-methyl guanosine (M1G), 2 '-O-methyl guanosine or N6,2' -O-dimethyl guanosine (M6 Am)), methoxyethoxy modifications (such as 2-methoxyethoxyadenosine, 2-methoxyethoxycytidine, 2-methoxyethoxyguanosine, 2-methoxyethoxyuridine), fluoridation modifications, pseudouracil modifications (ψ), or methyl pseudouracil modifications (M1- ψ), the cap structure may be cap0, cap1 or cap2 cap structures.
In the preparation method of the present invention, the buffer solution 2) may be selected from acetic acid/sodium acetate solution or citric acid/sodium citrate solution; citric acid/sodium citrate solution is preferred.
In a further preferred preparation method, the concentration of the acetic acid/sodium acetate solution or the citric acid/sodium citrate solution is 1mM-1M; more preferably 20mM-500mM; most preferably 100mM.
In a fourth aspect, the present invention provides the use of the feedstock composition for preparing antioxidant lipid nanoparticles according to the first aspect for preparing a vaccine or genetically modified immune cells, or for inducing reprogramming and redifferentiation of a plurality of primary cells' multifunctional stem cells (ipscs).
In a preferred application of the invention, the vaccine can be a tumor vaccine, a coronavirus vaccine, a monkey pox virus vaccine or a flavivirus vaccine, etc.
In a preferred application of the invention, the genetically modified immune cells are RNA-based gene transient overexpression systems or chimeric antigen receptor immune cells.
In the application of the invention, the antioxidant lipid nanoparticle prepared from the raw material composition can be used as a delivery body for introducing various exogenous nucleic acid molecules into cells, including DNA nucleic acid molecules and RNA nucleic acid molecules. DNA nucleic acid molecules such as plasmids, single-stranded DNA molecules and double-stranded DNA molecules. RNA nucleic acid molecules include protein-encoding linear RNAs, circular RNAs, self-replicating RNAs, and various non-coding RNAs such as microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs and long-chain non-coding RNAs. RNA molecules can be provided with various base modifications and/or cap structures, including but not limited to: methylation modifications (such as N6-methyl adenosine (M6A), N1-methyl adenosine (M1A), 5-methyl cytidine (M5C), 3-methyl cytidine (M3C), N7-methyl guanosine (M7G), 1-methyl guanosine (M1G), 2 '-O-methyl guanosine or N6,2' -O-dimethyl guanosine (M6 Am)), methoxyethoxy modifications (such as 2-methoxyethoxy adenosine, 2-methoxyethoxy cytidine, 2-methoxyethoxy guanosine or 2-methoxyethoxy uridine), fluoridation modifications, pseudouracil modifications (ψ) or methyl pseudouracil modifications (M1- ψ), the cap structure may be cap0, cap1 or cap2 cap structures.
Compared with the prior art, the invention can prepare the nucleic acid drug delivery system with oxidation-reduction resistance, local inflammatory reaction alleviation and mRNA vaccine expression efficiency promotion by optimizing the raw material components and proportion of LNP, and the system has sufficient stability, safety and higher loading efficiency, and can obviously improve the cell antigen presenting capability and organism immune response. The delivery system of the invention can also be popularized to the field of treatment of infectious disease vaccines and other diseases; and the raw materials are low in cost, and the method is suitable for large-scale production.
Drawings
FIG. 1 is a TEM photograph (scale bar 100 nm) of 7 mRNA lipid nanoparticles entrapped with OVA antigen prepared in example 1 and comparative example 1 of the present invention.
FIG. 2 shows the expression of GFP-mRNA 24 hours after transfection of mouse DC2.4 cells with LNP-mRNA and LNP (antioxidant) -mRNA in the experiment described in example 7 of the present invention.
FIG. 3 shows the results of antigen presentation 24 hours after transfection of mouse DC2.4 cells with LNP formulations with various antioxidants in the experiment described in example 8 of the present invention.
FIG. 4 shows the expression of Luci-mRNA after intramuscular injection of LNP formulations supplemented with various antioxidants in example 9 of the invention in mice.
FIG. 5 shows GFP-mRNA expression 24 hours after transfection of mouse DC2.4 cells with different proportions of VE added to LNP (VE) -mRNA in example 10 of the present invention.
FIG. 6 shows the results of antigen presentation 24 hours after transfection of mouse DC2.4 cells with LNP (VE) -mRNA nanoparticles added at different ratios of VE in example 11 of the present invention.
FIG. 7 shows the tumor volume change curves of LNP@mRNA and LNP (VE) -mRNA vaccines of example 12 of the present invention after treatment in a mouse melanoma model.
FIG. 8 shows the tumor therapeutic effect of LNP@mRNA and LNP (VE) -mRNA vaccines of example 12 of the present invention after treatment in a mouse melanoma model.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention constructs liposome nano-particles with antioxidation by using lipid components and antioxidants according to the following steps:
step (A1), mixing the ionizable lipid, the polyethylene glycol lipid, the neutral lipid, the steroid lipid and the antioxidant according to a certain proportion to obtain a raw material mixture, and dissolving the raw material mixture by using a solvent.
Step (A2) the nucleic acid is dissolved in a buffer solution of an appropriate pH.
Step (A3) mixing the liposome solution in step (A1) and the nucleic acid solution in step (A2), controlling the mixing volume ratio of the liposome solution and the nucleic acid solution and/or the mass ratio of the mixed raw material mixture and the nucleic acid, coextruding the aqueous phase solution and the organic phase solution by utilizing a microfluidic technology to prepare nucleic acid-entrapped LNPs, and then performing ultrafiltration or dialysis on the LNPs to obtain the nucleic acid-entrapped LNPs for in vivo delivery.
Preferably, the antioxidant used in step (A1) is vitamin a, vitamin C, vitamin E, coenzyme Q10, lipoic acid or polyphenol, or derivatives thereof; further preferred is vitamin E, coenzyme Q10 or lipoic acid, or derivatives thereof; more preferably vitamin E, lipoic acid or derivatives thereof; most preferred is vitamin E or a derivative thereof.
Preferably, the solvent used in step (A1) for dissolving the lipid molecules is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide or N, N-dimethylformamide; more preferably ethanol, tetrahydrofuran or acetone; most preferred is ethanol.
The present inventors have found through experiments that the mass ratio of LNP feed composition (i.e., antioxidant-containing lipids) to nucleic acid, the concentration of each lipid molecule in the organic phase, and the volume ratio of the organic phase to the aqueous phase all affect mRNA translation and expression, and thus, the above conditions were all screened and a range of conditions was obtained that gave the best transfection results.
Preferably, the proportion of ionizable lipids in step (A1) is 10% -70% (e.g. may be 10%, 20%, 30%, 40%, 50%, 60% or 70%) based on the total weight of the raw material mixture; more preferably 30% -60% (which may be, for example, 30%, 35%, 40%, 45%, 50%, 55% or 60%); further preferably 40% -55% (which may be, for example, 40%, 42%, 44%, 45%, 46%, 48% or 54%); most preferably 54%.
Preferably, the proportion of polyethylene glycol lipid in step (A1) is 1% -20% (e.g. may be 1%, 5%, 8%, 10%, 15%, 18% or 20%) based on the total weight of the raw material mixture; more preferably, the ratio is 5% -15% (e.g., may be 5%, 8%, 10%, 12%, 13%, 14% or 15%); further preferably 10% -15% (which may be 11%, 12%, 13% or 14%, for example); the most preferred ratio is 11%.
Preferably, the proportion of steroid lipids in step (A1) is 5% -60% (e.g. may be 5%, 10%, 20%, 30%, 40%, 50% or 60%) based on the total weight of the raw material mixture; more preferred ratios are 10% -50% (e.g., may be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%); further preferred ratios are 10% -30% (e.g. may be 10%, 12%, 15%, 18%, 20%, 24%, 26%, 28% or 30%); the most preferred ratio is 20%.
Preferably, the neutral lipid in step (A1) is present in a proportion of 1% -30% (e.g. may be 1%, 5%, 8%, 10%, 15%, 18%, 25% or 30%) based on the total weight of the raw material mixture; more preferably, the ratio is 5% -15% (e.g., may be 5%, 8%, 10%, 12% or 15%); further preferred proportions are 8% -10% (e.g. may be 8%, 9% or 10%); the optimal ratio is 10%.
Preferably, the antioxidant in step (A1) is vitamin E or a derivative thereof in a proportion of 1% -50% (e.g. may be 1%, 5%, 10%, 20%, 30%, 40%, 45% or 50%) by weight of the total weight of the raw material mixture; more preferably, the ratio is 2% -50% (e.g., may be 2%, 5%, 10%, 20%, 30%, 40%, 45% or 50%); further preferred ratios are 5% -30% (e.g. may be 5%, 10%, 20%, 30%, 40%, 45% or 50%); the most preferred ratio is 5%.
Preferably, the buffer solution in the step (A2) is acetic acid/sodium acetate solution, citric acid/sodium citrate solution; most preferred is a citric acid/sodium citrate solution.
Furthermore, the inventors have found through experiments that the pH of the buffer solution in which the nucleic acid molecules are dissolved needs to be adjusted to a suitable range, because when the aqueous phase is mixed with the organic phase, the ionizable lipid can be protonated in an acidic environment and positively charged, and thus can electrostatically interact with negatively charged RNA. Thus, the pH of the buffer solution in step (A2) may be 3-9 (e.g. may be 3, 4, 5, 6, 7, 8 or 9); but preferably the pH is 4-6 (e.g. may be 4, 5 or 6); the most preferred pH is 5.
Furthermore, the inventors found through experiments that the concentration of the buffer solution dissolving the nucleic acid molecules also affects the transfection efficiency of mRNA, and therefore, it is preferable that the concentration of the buffer solution in the step (A2) is 1mM-1M (for example, it may be 1mM, 50mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM or 1M); more preferably, the concentration is 20mM-500mM (e.g., 20mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, or 500 mM); the most preferred concentration is 100mM.
Preferably, the mass ratio of the mixed feedstock composition to nucleic acid in step (A3) is controlled to be 5:1-50:1 (e.g. may be 5:1, 10:1, 20:1, 30:1, 40:1 or 50:1); more preferably, the mass ratio is 10:1 to 30:1 (e.g., may be 10:1, 15:1, 20:1, 25:1, or 30:1); the most preferred mass ratio is 25:1.
Preferably, in step (A3), the volume ratio of liposome solution to nucleic acid solution is controlled to be 1:1-1:10; more preferably, the volume ratio is 1:1 to 1:5; the most preferred volume ratio is 1:3.
Example 1-example 6: encapsulating mRNA encoding OVA antigen with antioxidant lipid nanoparticle system
The ionizable lipid ALC-0315, neutral lipid DSPC, steroid lipid cholesterol, lipid-PEG ALC-0159 and antioxidant (6 antioxidants of VA, VC, VE, UQ, LA and PP respectively) are mixed and dissolved in ethanol solution according to the molar ratio of 46:10:35:2:7, so as to obtain 6 different organic phase solutions containing lipid and antioxidant. mRNA encoding OVA antigen was dissolved in sodium citrate (100 mM) buffer solution at pH 5.0 to give an aqueous solution containing nucleic acid molecules. Mixing each organic phase solution with the aqueous phase solution according to the volume ratio of 1:3, and controlling the mass of the lipid contained in the mixture and mRNA coding the OVA antigen to be 25:1 to obtain white emulsion. And then the white emulsion is co-extruded by utilizing a microfluidic technology to prepare spherical particles. Ethanol was then removed by dialysis to give 6 LNP (antioxidant) -mRNAs harboring mRNAs encoding OVA antigen, corresponding to examples 1-6, respectively designated LNP (VA) -mRNAs, LNP (VC) -mRNAs, LNP (VE) -mRNAs, LNP (coenzyme Q10) -mRNAs, LNP (lipoic acid-NH) 2 ) -mRNA and LNP (polyphenol) -mRNA.
Particle size distribution characterization and morphology characterization were performed on the 6 LNPs obtained in examples 1-6 using Transmission Electron Microscopy (TEM). TEM experiment results show that the LNP (antioxidant) -mRNA particles are spherical and have a particle size of about 50-100nm, as shown in FIG. 1.
Comparative example 1: encapsulation of mRNA encoding OVA antigen using common lipid nanoparticle systems
The same ionizable lipid ALC-0315, neutral lipid DSPC, steroid lipid cholesterol and lipid-PEG ALC-0159 as in example one were mixed in a molar ratio of 45:10:42:3 to obtain an organic phase solution containing lipid and no antioxidant, and an aqueous phase solution was prepared in the same manner as in example one and mixed in a volume ratio of 1:3 and co-extruded to obtain LNP-mRNA in which mRNA encoding OVA antigen was entrapped. Particle size distribution characterization and morphology characterization were performed on the obtained LNPs using Transmission Electron Microscopy (TEM). TEM experiment results show that LNP-mRNA has spherical shape and particle size of 50-100nm.
Example 7: antioxidant lipid nanoparticle systems entrap mRNA encoding GFP for cell transfection
To evaluate the effect of the invention on LNP transfection efficiency after the addition of various antioxidants, LNP-mRNA and LNP (antioxidant) -mRNA were obtained as described in example 1-example 6 and comparative example 1, 2X 10 per 1. Mu.g RNA transfection 6 Cells, LNP-mRNA nanoparticles of comparative example 1 and 6 LNP (antioxidant) -mrnas were added to a random group of mouse DC2.4 cell lines, respectively, and after culturing for 24 hours, cells were collected and the transfection efficiency of the different LNPs on DC cells was analyzed by flow cytometry. The results are shown in FIG. 2: the LNP (antioxidant) -mRNA prepared in examples 1-6 of the present invention significantly enhanced expression and transfection of mRNA in DC cells compared to the LNP-mRNA prepared in comparative example 1.
Example 8: mRNA encoding OVA antigen entrapped by antioxidant lipid nanoparticle system is used for cell antigen presenting experiment
In view of the excellent performance of antioxidants in cell transfection exhibited by example 7, we continued to explore the antigen presenting efficiency of LNP (antioxidant) -mRNA of examples 1, 2, 3, 4, 6 (i.e., LNP with VA, VC, VE, UQ and PP added to lipid nanoparticles, respectively). LNP-mRNA was obtained as described in comparative example 1, and LNP (VA) -mRNA, LNP (VC) -mRNA, LNP (VE) -mRNA, LNP (UQ) -mRNA and LNP (PP) -mRNA were transfected 2X 10 per 1. Mu.g of RNA as described in examples 1, 2, 3, 4 and 6 6 Cells the 6 lipid nanoparticles prepared above were added to a random group of mouse DC2.4 cell lines, respectively, and after culturing for 24 hours, cells were collected, stained with APC channel anti-mouse H-2Kb bound to SIINFEKL antibody (biolegend), washed and fixed with 4% paraformaldehyde, and each group of DC cell antigen presenting efficiency was analyzed using a flow cytometer. The results are shown in FIG. 3: LNP (VA) -mRNA, LNP (VE) -mRNA and LNP UQ) -mRNA significantly enhanced the antigen presenting ability of DC cells compared to LNP-mRNA.
Example 9: antioxidant lipid nanoparticle system entrapping Luci-mRNA for in vivo RNA expression verification
LNP-mRNA was obtained as described in comparative example 1, the 6 LNP (antioxidant) -mRNA was prepared as described in example 1-example 6, and randomly grouped mice were given intramuscular injections with 7 LNP lipid nanoparticles per 2. Mu.g RNA/mouse, respectively, and whole body fluorescence imaging was performed for each group of mice using three-dimensional in vivo imaging for 24 hours, and the fluorescence expression of Lucifarase at the local injection site was measured. The results are shown in FIG. 4: compared with LNP-mRNA, each LNP (antioxidant) -mRNA can enhance the expression of Luci-mRNA in mice, wherein the enhancement effect of LNP (VA) -mRNA, LNP (VE) -mRNA, LNP (UQ) -mRNA and LNP (LA) -mRNA is obvious, and particularly the enhancement effect of LNP (VE) -mRNA is most obvious.
Example 10: effect of addition of VE in different ratios on cell transfection efficiency
In view of the excellent performance of the LNP constructed as an antioxidant by VE in various indices in vitro and in vivo, we continued to explore the effect of adding different proportions of VE on its GFP transfection efficiency. LNP-mRNA (GFP) (denoted "LNP@mRNA") was obtained as described in comparative example 1, and VE was added to the LNP starting material in various proportions (molar ratios) in the procedure of reference example 3 to obtain LNP (VE) -mRNA (GFP) containing various levels of VE, denoted "LNP (VE 1.7%) @ mRNA", "LNP (VE 3.5%) @ mRNA", "LNP (VE 7%) @ mRNA" and "LNP (VE 10.5%) @ mRNA", respectively, each 1. Mu.g of RNA was transfected 2X 10 @ mRNA @ 6 Cells, lnp@mrna nanoparticles, LNP (VE 1.7%) @ mRNA, LNP (VE 3.5%) @ mRNA, LNP (VE 7%) @ mRNA, and LNP (VE 10.5%) @ mRNA were added to a random group of mouse DC2.4 cell lines, respectively, and after culturing for 24 hours, cells were collected and each group of LNP transfection efficiency on DC cells was analyzed by flow cytometry. The results are shown in FIG. 5: compared with LNP@mRNA, the LNP (VE) -mRNA with various VE contents can obviously improve GFP expression efficiency, and especially the LNP (VE 7%) @mRNA added with 7% VE (molar ratio) shows the highest GFP expression efficiency.
Example 11 Effect of addition of different proportions of VE on DC antigen presentation efficiency
In view of the excellent performance of different proportions of VE in cell transfection, we continued to explore the antigen presenting efficiency of VE-added LNP. LNP-mRNA was obtained as described in comparative example 1, with reference to LNP (VE) -mRNA (designated "LNP@mRNA"), and with reference to example 3, VE was added to the LNP feedstock in varying proportions to produce LNP (VE) -mRNA (GFP) containing varying levels of VE, designated "LNP (VE 1.7%) @ mRNA", "LNP (VE 3.5%) @ mRNA", "LNP (VE 7%) @ mRNA" and "LNP (VE 10.5%) @ mRNA", respectively, transfected 2X 10 per 1. Mu.g RNA 6 Cells lnp@mrna, LNP (VE 1.7%) @mrna, LNP (VE 3.5%) @mrna, LNP (VE 7%) @mrna and LNP (VE 10.5%) @mrna were added to a random grouping of mouse DC2.4 cell lines, after 24 hours of culture, cells were collected, stained with APC channel anti-mouse H-2Kb bound to SIINFEKL antibody (biolegend), washed and fixed with 4% paraformaldehyde, and each group of DC cell antigen presenting efficiencies were analyzed using a flow cytometer. The results are shown in FIG. 6: compared with LNP@mRNA, the LNP (VE) -mRNA with various VE contents can obviously improve the antigen presenting capability of DC cells, and especially the LNP (VE) -mRNA added with 7% VE shows the optimal antigen presenting capability of DC cells.
Example 12: mRNA encoding OVA antigen entrapped by antioxidant lipid nanoparticle system for mouse antitumor vaccine
In view of the in vitro and in vivo screening results of the above system, the antioxidant lipid nanoparticle added with 7 mole percent VE in all the antioxidant-added formulations of the present invention has optimal cell transfection effect and antigen presenting capability. Therefore, in the anti-tumor efficacy verification experiment of living bodies, VE is used as an anti-oxidation component to construct anti-oxidation lipid nano particles to evaluate the anti-tumor efficacy. C57/B6J mice were selected and injected subcutaneously 5X 10 5 Melanoma B16 cell line expressing chicken ovalbumin OVA (257-264) antigen peptide after nine days of tumor growth, mice were randomly grouped, LNP-mRNA (designated LNP@mRNA vaccine) was obtained as described in comparative example 1, LNP (VE) -mRNA vaccine supplemented with 7% molar percent VE was prepared as described in reference example 3 byIntramuscular injection was performed by inoculating each group of mice with the two vaccines and PBS. The vaccine and PBS were again given at the same dose on the fifteenth day, mice (4 per group) were sacrificed on the twenty-third day, tumor tissues were taken, and photographed for analysis. The three groups of tumor treatment effects are shown in fig. 7 and 8: the tumor treatment effect of the treatment group inoculated with LNP (VE) -mRNA is obviously better than that of the treatment group inoculated with LNP@mRNA and PBS, and the average tumor volume of 4 mice has almost no obvious change in the whole treatment period; whereas the mean tumor volume of mice vaccinated with lnp@mrna and PBS group was significantly increased.
The experiment proves that the lipid nanoparticle coated with the nucleic acid molecules can obviously improve the immune activation effect in mice and the anti-tumor curative effect.

Claims (26)

1. A raw material composition for preparing antioxidant lipid nanoparticles, characterized in that its components comprise ionizable lipids, neutral lipids, polyethylene glycol lipids, steroidal lipids and antioxidants; the weight portions of the components are as follows: 10-70 parts of ionized lipid, 1-30 parts of neutral lipid, 1-20 parts of polyethylene glycol lipid, 5-60 parts of steroid lipid and 1-50 parts of antioxidant; the preferred proportions of the components are as follows: 30-60 parts of ionizable lipid, 5-15 parts of neutral lipid, 1-15 parts of polyethylene glycol lipid, 10-50 parts of steroid lipid and 2-50 parts of antioxidant; more preferred ratios of the components are as follows: 40-55 parts of ionizable lipid, 8-10 parts of neutral lipid, 5-15 parts of polyethylene glycol lipid, 10-30 parts of steroid lipid and 5-30 parts of antioxidant; the most preferred ratios of the components are as follows: 54 parts of ionizable lipid, 10 parts of neutral lipid, 11 parts of polyethylene glycol lipid, 20 parts of steroid lipid and 5 parts of antioxidant.
2. The feedstock composition according to claim 1, wherein the antioxidant is selected from any one of the following: vitamin A (VA) or a derivative thereof, vitamin C (VC) or a derivative thereof, vitamin E (VE) or a derivative thereof, coenzyme Q10 (UQ) or a derivative thereof, lipoic Acid (LA) or a derivative thereof, or polyphenol (PP) or a derivative thereof, or a mixture of any two or more thereof; preferably vitamin E or a derivative thereof, coenzyme Q10 or a derivative thereof or lipoic acid or a derivative thereof; further preferred is vitamin E or a derivative thereof, or lipoic acid or a derivative thereof; most preferred is vitamin E or a derivative thereof.
3. The feedstock composition according to claim 1, wherein the ionizable lipid is selected from the group consisting of lipid molecules represented by any one of the following lipid frameworks comprising one or more ionizable sites: c12-200, ALC-0315, SM102, DODAP, DODMA, DOBAQ, YSK05, dlin-DMA, dlin-KC2-DMA or Dlin-MC3-DMA, or a mixture of any two or more thereof.
4. The feedstock composition according to claim 1, wherein the neutral lipid is selected from the group consisting of any one or more of the following: 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), or 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
5. The feedstock composition according to claim 1, wherein the polyethylene glycol lipid is selected from the group consisting of any one or more of the following: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerideamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxy propyl-3-amine (PEG-c-DMA).
6. The composition of matter of claim 1, wherein said steroid lipid is selected from the group consisting of any one or a mixture of two or more of the following: oat sterols, beta-sitosterols, campesterols, ergocalcitols, campesterols, cholestanol, cholesterol, fecal sterols, dehydrocholesterol, desmosterols, dihydroergocalcitols, dihydrocholesterol, dihydroergosterols, black-sea sterols, episterols, ergosterols, fucosterols, hexahydrophotopterols, hydroxycholesterols; lanosterol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, or lithocholic acid.
7. A nucleic acid drug delivery system is spherical particles with the particle diameter of 50-100nm, which are formed by entrapping nucleic acid molecules in LNP; wherein the LNP comprises, by weight, 10-70 parts of ionizable lipid, 10-30 parts of neutral lipid, 1-20 parts of polyethylene glycol lipid, 5-60 parts of steroid lipid and 1-50 parts of antioxidant; the LNP comprises, by weight, 30-60 parts of ionizable lipid, 5-15 parts of neutral lipid, 1-15 parts of polyethylene glycol lipid, 10-50 parts of steroid lipid and 2-50 parts of antioxidant; more preferred LNP materials, by weight, are composed of 40-55 parts of ionizable lipids, 8-10 parts of neutral lipids, 5-15 parts of polyethylene glycol lipids, 10-30 parts of steroid lipids and 5-30 parts of antioxidants; further preferred LNP materials consist of, in parts by weight, 54 parts of ionizable lipids, 10 parts of neutral lipids, 11 parts of polyethylene glycol lipids, 20 parts of steroid lipids and 5 parts of antioxidants; the weight ratio of LNP to nucleic acid molecule is 5:1-50:1; preferably 10:1 to 30:1; most preferably 25:1.
8. The delivery system of claim 7, wherein the antioxidant is Vitamin A (VA) or a derivative thereof, vitamin C (VC) or a derivative thereof, vitamin E (VE) or a derivative thereof, coenzyme Q10 (UQ) or a derivative thereof, lipoic Acid (LA) or a derivative thereof, or Polyphenols PP) or a derivative thereof.
9. The delivery system of claim 7, wherein said ionizable lipid is selected from the group consisting of lipid molecules represented by a lipid backbone containing one or more ionizable sites: any one or more of C12-200, ALC-0315, SM102, DODAP, DODMA, DOBAQ, YSK05, dlin-DMA, dlin-KC2-DMA or Dlin-MC 3-DMA.
10. The delivery system of claim 7, wherein said polyethylene glycol lipid is selected from the group consisting of: any one or more than two of 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerideamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).
11. The delivery system of claim 7, wherein said neutral lipid is selected from the group consisting of: any one or a combination of more than two of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycero) (DOPG), oleoyl phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
12. The delivery system of claim 7, wherein said steroid lipid is selected from the group consisting of: oat sterols, beta-sitosterols, campesterols, ergocalcitols, campesterols, cholestanol, cholesterol, fecal sterols, dehydrocholesterol, desmosterols, dihydroergocalcitols, dihydrocholesterol, dihydroergosterols, black-sea sterols, episterols, ergosterols, fucosterols, hexahydrophotopterols, hydroxycholesterols; lanosterol, photosterol, sitosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid.
13. The delivery system of claim 7, wherein the nucleic acid molecule is a DNA nucleic acid molecule or an RNA nucleic acid molecule; in a preferred embodiment, the DNA nucleic acid molecule is a plasmid, a single-stranded DNA molecule or a double-stranded DNA molecule; in a preferred embodiment, the RNA nucleic acid molecule is a protein-encoding linear RNA, a protein-encoding circular RNA, a self-replicating RNA or various non-encoding RNAs, more preferably microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs or long-chain non-encoding RNAs; in a preferred embodiment, the RNA molecule has various base modifications and/or cap structures, and the base modifications are further preferred: methylation modifications (such as N6-methyl adenosine (M6A), N1-methyl adenosine (M1A), 5-methyl cytidine (M5C), 3-methyl cytidine (M3C), N7-methyl guanosine (M7G) or 1-methyl guanosine (M1G), 2 '-O-methyl guanosine or N6,2' -O-dimethyl guanosine (M6 Am)), methoxyethoxy modifications (such as 2-methoxyethoxyadenosine, 2-methoxyethoxycytidine, 2-methoxyethoxyguanosine, 2-methoxyethoxyuridine), fluoridation modifications, pseudouracil modifications (ψ), or methyl pseudouracil modifications (M1- ψ), with cap structures of cap0, cap1 or cap2 being further preferred.
14. A method of preparing the nucleic acid drug delivery system of any one of claims 7-13, comprising:
1) Dissolving a mixture of 10-70% of an ionizable lipid, 1% -30% of a neutral lipid, 1% -20% of a polyethylene glycol lipid, 5% -60% of a steroid lipid and 1% -50% of an antioxidant, preferably a mixture of 30% -60% of an ionizable lipid, 5% -15% of a neutral lipid, 1% -15% of a polyethylene glycol lipid, 10% -50% of a steroid lipid and 2% -50% of an antioxidant, more preferably a mixture of 40% -55% of an ionizable lipid, 8% -10% of a neutral lipid, 5% -15% of a polyethylene glycol lipid, 10% -30% of a steroid lipid and 5% -30% of an antioxidant, most preferably a mixture of 54% of an ionizable lipid, 10% of a neutral lipid, 11% of a polyethylene glycol lipid, 20% of a steroid lipid and 5% of an antioxidant in an organic solvent to obtain an antioxidant-containing lipid solution;
2) Dissolving nucleic acid in a buffer solution having a pH of 3 to 9, preferably in a buffer solution having a pH of 4 to 6, more preferably in a buffer solution having a pH of 5, to obtain a nucleic acid solution;
3) Mixing the lipid solution obtained in the step 1) with the nucleic acid solution obtained in the step 2), and controlling the weight ratio of the total lipid containing the antioxidant to the nucleic acid molecules to be 5:1-50:1 after mixing; and then extruding the mixed solution by utilizing a microfluidic technology to prepare spherical particles, namely the LNPs for coating the nucleic acid molecules.
15. The method of claim 14, wherein the antioxidant of 1) is Vitamin A (VA) or a derivative thereof, vitamin C (VC) or a derivative thereof, vitamin E (VE) or a derivative thereof, coenzyme Q10 (UQ) or a derivative thereof, lipoic Acid (LA) or a derivative thereof, or Polyphenols PP) or a derivative thereof.
16. The method of claim 14, wherein 1) the ionizable lipid is selected from the group consisting of lipid molecules represented by a lipid backbone containing one or more ionizable sites: any one or more of C12-200, ALC-0315, SM102, DODAP, DODMA, DOBAQ, YSK05, dlin-DMA, dlin-KC2-DMA or Dlin-MC 3-DMA.
17. The method of claim 14, wherein 1) the polyethylene glycol lipid is selected from the group consisting of: any one or more than two of 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerideamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).
18. The method of claim 14, wherein 1) the neutral lipid is selected from the group consisting of: any one or a combination of more than two of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycero) (DOPG), oleoyl phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE).
19. The method of claim 14, wherein 1) the steroid lipid is selected from the group consisting of: oat sterols, beta-sitosterols, campesterols, ergocalcitols, campesterols, cholestanol, cholesterol, fecal sterols, dehydrocholesterol, desmosterols, dihydroergocalcitols, dihydrocholesterol, dihydroergosterols, black-sea sterols, episterols, ergosterols, fucosterols, hexahydrophotopterols, hydroxycholesterols; lanosterol, photosterol, sitosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid.
20. The method of claim 14, wherein 1) the organic solvent is selected from the group consisting of methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, and N, N-dimethylformamide; preferably ethanol, tetrahydrofuran or acetone; most preferred is ethanol.
21. The method of claim 14, wherein 2) the nucleic acid molecule is a DNA nucleic acid molecule or an RNA nucleic acid molecule; in a preferred embodiment, the DNA nucleic acid molecule is a plasmid, a single-stranded DNA molecule or a double-stranded DNA molecule; in a preferred embodiment, the RNA nucleic acid molecule is a protein-encoding linear RNA, a protein-encoding circular RNA, a self-replicating RNA or various non-encoding RNAs, more preferably microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs or long-chain non-encoding RNAs; in a preferred embodiment, the RNA molecule has various base modifications and/or cap structures, and the base modifications are further preferred: methylation modifications (such as N6-methyl adenosine (M6A), N1-methyl adenosine (M1A), 5-methyl cytidine (M5C), 3-methyl cytidine (M3C), N7-methyl guanosine (M7G) or 1-methyl guanosine (M1G), 2 '-O-methyl guanosine or N6,2' -O-dimethyl guanosine (M6 Am)), methoxyethoxy modifications (such as 2-methoxyethoxyadenosine, 2-methoxyethoxycytidine, 2-methoxyethoxyguanosine, 2-methoxyethoxyuridine), fluoridation modifications, pseudouracil modifications (ψ), or methyl pseudouracil modifications (M1- ψ), with cap structures of cap0, cap1 or cap2 being further preferred.
22. The method of claim 14, wherein 2) the buffer solution is selected from the group consisting of acetic acid/sodium acetate solution or citric acid/sodium citrate solution; citric acid/sodium citrate solution is preferred.
23. The method of claim 23, wherein the concentration of the acetic acid/sodium acetate solution or the citric acid/sodium citrate solution is 1mM to 1M; more preferably 20mM-500mM; most preferably 100mM.
24. Use of the raw material composition for preparing antioxidant lipid nanoparticles according to any one of claims 1-6 for preparing vaccines or genetically modified immune cells, or for inducing reprogramming and redifferentiation of multifunctional stem cells (ipscs) of a plurality of primary cells.
25. The use according to claim 24, wherein the vaccine is a tumor vaccine, a coronavirus vaccine, a monkey pox virus vaccine or a flavivirus vaccine.
26. The use of claim 24, wherein the genetically modified immune cell is an RNA-based gene transient overexpression system or a chimeric antigen receptor immune cell.
CN202311211551.8A 2023-09-20 2023-09-20 Nucleic acid-entrapped antioxidant lipid nanoparticle, and preparation method and application thereof Pending CN117257761A (en)

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