CN114456081A - 一种可离子化脂质及其制备方法和应用 - Google Patents
一种可离子化脂质及其制备方法和应用 Download PDFInfo
- Publication number
- CN114456081A CN114456081A CN202210137654.3A CN202210137654A CN114456081A CN 114456081 A CN114456081 A CN 114456081A CN 202210137654 A CN202210137654 A CN 202210137654A CN 114456081 A CN114456081 A CN 114456081A
- Authority
- CN
- China
- Prior art keywords
- lipid
- mrna
- delivery system
- solution
- ionizable lipid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 150000002632 lipids Chemical class 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 69
- 239000000243 solution Substances 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 13
- 150000003904 phospholipids Chemical class 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000006185 dispersion Substances 0.000 claims description 5
- 238000006482 condensation reaction Methods 0.000 claims description 4
- 238000005886 esterification reaction Methods 0.000 claims description 4
- NVRVNSHHLPQGCU-UHFFFAOYSA-N 6-bromohexanoic acid Chemical compound OC(=O)CCCCCBr NVRVNSHHLPQGCU-UHFFFAOYSA-N 0.000 claims description 3
- BKJFDZSBZWHRNH-UHFFFAOYSA-N 8-bromooctanoic acid Chemical compound OC(=O)CCCCCCCBr BKJFDZSBZWHRNH-UHFFFAOYSA-N 0.000 claims description 3
- -1 9-heptadecyl Chemical group 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- KJIOQYGWTQBHNH-UHFFFAOYSA-N undecanol Chemical compound CCCCCCCCCCCO KJIOQYGWTQBHNH-UHFFFAOYSA-N 0.000 claims description 3
- KLSJWNVTNUYHDU-UHFFFAOYSA-N Amitrole Chemical compound NC1=NC=NN1 KLSJWNVTNUYHDU-UHFFFAOYSA-N 0.000 claims description 2
- 238000005538 encapsulation Methods 0.000 abstract description 6
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 5
- 239000001257 hydrogen Substances 0.000 abstract description 5
- 230000002378 acidificating effect Effects 0.000 abstract description 4
- 125000000217 alkyl group Chemical group 0.000 abstract description 4
- 230000001808 coupling effect Effects 0.000 abstract description 4
- 229910052736 halogen Inorganic materials 0.000 abstract description 4
- 150000002367 halogens Chemical class 0.000 abstract description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 abstract description 4
- 241000699670 Mus sp. Species 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 10
- 239000002502 liposome Substances 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 239000013603 viral vector Substances 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 125000002091 cationic group Chemical group 0.000 description 5
- 239000002105 nanoparticle Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000004809 thin layer chromatography Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 241000021375 Xenogenes Species 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- WTWWTKPAEZQYPW-UHFFFAOYSA-N heptadecan-9-ol Chemical compound CCCCCCCCC(O)CCCCCCCC WTWWTKPAEZQYPW-UHFFFAOYSA-N 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
- JFBCSFJKETUREV-LJAQVGFWSA-N 1,2-ditetradecanoyl-sn-glycerol Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](CO)OC(=O)CCCCCCCCCCCCC JFBCSFJKETUREV-LJAQVGFWSA-N 0.000 description 1
- RISHBQWFBUTROQ-UHFFFAOYSA-N 3-amino-1,1,1-trifluoropropan-2-ol Chemical compound NCC(O)C(F)(F)F RISHBQWFBUTROQ-UHFFFAOYSA-N 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 1
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 1
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 102000044890 human EPO Human genes 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229940038694 mRNA-based vaccine Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1816—Erythropoietin [EPO]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
Abstract
本发明公开了一种可离子化脂质及其制备方法和应用，所述可离子化脂质的结构式为
本发明上述可离子化脂质在酸性条件下，带正电，可以与mRNA的负电磷酸基团静电吸引，含卤素的烷基能够与膜具有更好的亲和性，同时，具有的羟基结构易于和mRNA的碱基形成氢键，从而达到很好的耦合效果，具有较高的包封效率。
Description
技术领域
本发明涉及mRNA递送技术领域，具体涉及一种可离子化脂质及其制备方法和应用。
背景技术
治疗性核酸包括信使RNA(mRNA)、小干扰RNA(siRNA)、微RNA(miRNA)、反义寡核苷酸、核酶、质粒和免疫刺激性核酸。此类核酸通过多种机制起作用。对于mRNA，mRNA是蛋白质编码DNA翻译和细胞质中核糖体产生蛋白质之间的中间步骤。两种主要类型的mRNA目前被研究作为疫苗：非复制的mRNA和病毒衍生的、自扩增的mRNA。传统的基于mRNA的疫苗编码相关的抗原，并含有5′和3′非翻译区(UTRs)，而自扩增mRNA不仅编码抗原，还编码病毒复制机制，使胞内RNA的自扩增和蛋白质的高表达成为可能。
mRNA的治疗性应用极其广泛，因为mRNA是非感染性、非整合性平台，不存在感染或插入突变的潜在风险。此外，mRNA能够被正常的细胞过程降解，其体内半衰期可以通过使用各种修饰和递送方法来调节。该基因固有的免疫原性可以下调，以进一步提高安全性。而各种类型的修饰使mRNA更加稳定，具有高度可翻译性。mRNA是最小的遗传载体；因此，避免了抗载体免疫，mRNA可以重复给药。迄今为止，mRNA构建体在体外和体内模型中都已显示出高效的蛋白质表达。
然而，裸露的mRNA存在的问题是：首先，mRNA会被细胞外的RNA酶快速降解；其次，mRNA难以被有效内化(mRNA的高阴离子电荷密度、大小和亲水性阻止了其在细胞膜上有意义的被动扩散)；最后，裸露的mRNA可能存在着潜在的免疫原性。
穿过细胞膜并定位到适当的亚细胞区室一直是基于mRNA治疗的主要障碍。用于促进细胞对mRNA的摄取并保护其不被降解，且免疫原性较低的递送系统被开发出来。
用做mRNA递送系统的主要分为病毒载体及非病毒载体。目前病毒载体面临的两个问题是：首先病毒载体研发周期长，成本较高；其次病毒载体免疫原性较高，易产生较强烈的免疫反应，且病毒载体存在着诱导机体癌变的风险。
而非病毒载体递送mRNA的方式主要为脂质体、树突细胞、无机纳米粒子、阳离子细胞穿膜肽等。其中脂质体纳米颗粒结构具有诸多优势：首先结构为球形囊泡，可将mRNA包裹在内，抵御核酸酶的作用；第二，外表类似于细胞膜，易与受体细胞融合，转染效率高；第三，理论上，可递送不同大小片段的mRNA；第四，脂质体作为递送载体一般不受宿主限制。
目前市场上用做mRNA递送系统的主要为阳离子脂质体。阳离子脂质体面临的问题是：首先，带正电的阳离子脂质体易与血清中的蛋白质发生吸附，造成聚集以及被免疫细胞吞噬并清除；其次，短的循环半衰期以及与带负电荷的细胞和细胞外成分的非特异性结合。组织清除率低，不能循环给药，对慢性病疗效较差。而且阳离子脂质载体无法有效递送分子量大，结构复杂的自扩增mRNA。
发明内容
本发明的目的在于提供一种可离子化脂质及其制备方法和应用，本发明可离子化脂质在酸性条件下，带正电，可以与扩增mRNA的负电磷酸基团静电吸引，含卤素的烷基能够与膜具有更好的亲和性，同时，具有的羟基结构易于和mRNA的碱基形成氢键，从而达到很好的耦合效果，具有较高的包封效率。
为了实现本发明的上述目的，特采用以下技术方案：
本发明第一方面提供一种可离子化脂质，所述可离子化脂质的结构式如式I所示：
本发明上述可离子化脂质在酸性条件下，带正电，可以与扩增mRNA的负电磷酸基团静电吸引，含卤素的烷基能够与膜具有更好的亲和性，同时，具有的羟基结构易于和mRNA的碱基形成氢键，从而达到很好的耦合效果，具有较高的包封效率。
本发明第二方面提供一种上述可离子化脂质的制备方法，所述制备方法包括如下步骤：
(a)将十一醇和6-溴己酸进行酯化反应，得到中间体1；
(b)将9-十七醇和8-溴辛酸进行酯化反应，得到中间体2；
(c)将中间体2和3-氨基-1，1，1-三氟丙烷-2-醇进行缩合反应，得到中间体3；
(d)将中间体3和中间体1进行缩合反应，得到所述可离子化脂质。
本发明第三方面提供一种上述可离子化脂质在制备mRNA递送体系中的应用。
本发明第四方面提供一种mRNA递送体系，该mRNA递送体系包括上述可离子化脂质、磷脂、PEG脂质和结构性脂质和mRNA。
优选地，所述可离子化脂质、结构性脂质、磷脂和PEG脂质的摩尔比为50∶(35～40)∶(9～11)∶(1.3～1.8)。
优选地，所述可离子化脂质与mRNA的摩尔比为(8～15)∶1。
优选地，磷脂为DOPE或DSPC；PEG脂质为1，2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇，又称为PEG-DMG；结构性脂质为胆固醇。
本发明第五方面提供一种上述mRNA递送体系的制备方法，所述制备方法包括如下步骤：
(a)按照摩尔比，将可离子化脂质、结构性脂质、磷脂和PEG脂质溶于乙醇，得到脂质溶液；
(b)在微流控系统中，将脂质溶液以2～18mL/min的流速注入mRNA溶液中，得到纳米脂质分散液，经0.22μm无菌过滤器过滤后，得到所述mRNA递送体系。
优选地，所述mRNA溶液的浓度为0.08～0.2mg/mL，溶剂为pH值为3～4、50mM的柠檬酸钠缓冲液。
优选地，所述mRNA溶液和脂质溶液的体积比为(2～4)∶1。
与现有技术相比，本发明的有益效果至少包括：
本发明上述可离子化脂质在酸性条件下，带正电，可以与扩增mRNA的负电磷酸基团静电吸引，含卤素的烷基能够与膜具有更好的亲和性，同时，具有的羟基结构易于和mRNA的碱基形成氢键，从而达到很好的耦合效果，具有较高的包封效率。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。在所有附图中，类似的元件或部分一般由类似的附图标记标识。附图中，各元件或部分并不一定按照实际的比例绘制。
图1为本发明可离子化脂质的合成工艺；
图2为本发明可离子化脂质的1HNMR谱图；
图3为本发明实施例2制备的递送体系的粒径和Zeta电位图；
图4为测定游离mRNA的标准曲线；
图5为测定总mRNA的标准曲线；
图6为实验组小鼠6小时肌肉注射Fluc-mRNA药物后整体的化学发光强度数值；
图7为用IVIS活体成像系统读取实验组小鼠6小时肌肉注射Fluc-mRNA药物后整体化学发光信号；
图8为实验组小鼠6小时静脉注射Fluc-mRNA药物后各器官的化学发光强度数值；
图9为用IVIS活体成像系统读取的6小时静脉注射Fluc-mRNA药物后各器官的化学发光信号；
图10为6小时下静脉注射小鼠血清中hEPO-mRNA的浓度数值。
具体实施方式
下面将结合实施例对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只作为示例，而不能以此来限制本发明的保护范围。
需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
实施例1
本实施例为一种可离子化脂质的制备方法，该可离子化脂质的合成工艺如图1所示，该制备方法包括如下步骤：
(a)合成中间体1：
在0℃，氮气保护的条件下，向十一醇(2.27g，13.2mmol)、6-溴己酸(3.09g，15.8mmol)和DMAP(0.33g，2.64mmol)的二氯甲烷中溶液中添加EDCI(3.79g，19.8mmol)粉末，室温搅拌反应过夜，TLC显示反应结束，用2M盐酸酸化，用乙腈：正己烷＝1：1的萃取液进行萃取，干燥脱溶得到中间体1，计算收率为95％，不进行纯化直接用于下一步反应；
(b)合成中间体2：
在0℃，氮气保护的条件下，向9-十七醇(2.94g，13.2mmol)、8-溴辛酸(4.06g，15.8mmol)和DMAP(0.33g，2.64mmol)的二氯甲烷的溶液中添加EDCI(3.79g，19.8mmol)粉末，室温搅拌反应过夜，TLC显示反应结束，用2M盐酸酸化，用乙腈：正己烷＝1：1的萃取液进行萃取，干燥脱溶得到中间体2，计算收率93％，不进行纯化直接用于下一步反应；
(c)合成中间体3：
在室温下，将中间体2(0.23g，0.5mmol)和3-氨基-1，1，1-三氟丙烷-2-醇(0.066g0.75mmol)在异丙醇中搅拌过夜，反应温度70℃，TLC显示反应结束，粗品通过色谱纯化得到中间体3，纯化梯度：DCM/MeOH＝10:0～10:1，计算收率为52％；
(d)合成可离子化脂质(记为HK-01)
在氮气保护的条件下，向中间体3(0.67g，1.31mmol)在DMF中的溶液中加入中间体1(0.54g，1.57mmol)、碳酸钾(0.54g，3.93mmol)、碘化钾(0.002g，0.013mmol)；氮气保护80℃反应16h，TLC监测反应进程反应结束后，用水稀释有机相，用乙酸乙酯萃取，干燥脱溶，粗品通过色谱纯化得到可离子化脂HK-01，纯化梯度：DCM/MeOH＝10:0～10:2，计算收率为77％；
对可离子化脂质进行氢谱分析，分析结果如图2所示，
由图2可知，1HNMR(500MHz，CDCl3)δ4.86(p，J＝6.3Hz，1H)，4.10-3.97(m，1H)，2.98(dd，J＝12.9，6.8Hz，2H)，2.88(dd，J＝12.9，4.6Hz，1H)，2.75-2.59(m，2H)，2.34-2.21(m，2H)，1.60(dd，J＝19.8，13.1Hz，3H)，1.56-1.43(m，14H)，1.28(dt，J＝45.1，19.4Hz，50H)，0.93-0.77(m，10H)。
由图2可知，制备得到的可离子化脂结构如式I所示。
实施例2
本实施例为一种mRNA递送体系的制备方法，该制备方法包括如下步骤：
(a)按照摩尔比为50∶38.5∶10∶1.5，将可离子化脂质、结构性脂质、磷脂和PEG脂质溶于乙醇，得到脂质溶液；
(b)在微流控系统中，将脂质溶液以10mL/min的流速注入mRNA溶液中，得到纳米脂质分散液，经0.22μm无菌过滤器过滤后，得到mRNA递送体系，
其中，mRNA溶液的浓度为0.1mg/mL，溶剂为pH值为3.5、50mM的柠檬酸钠缓冲液，mRNA溶液和脂质溶液的体积比为3∶1，可离子化脂质与mRNA的摩尔比为10∶1；mRNA为trilink公司商品化的CleanCap mRNA。
使用Zetasizer Nano ZS(Malvern Instru ments Ltd，Malvern，Worcestershire，UK)测定上述制备得到的mRNA递送体系的粒度、多分散指数(PDI)和ζ电势，粒度是在1×PBS中测定且ζ电势是在15mM PBS中测定的；其中，粒径和电位测试结果如图3所示，
由图3可知，本发明递送体系的粒径为93.55nm，Zeta电位为-3.4mV。
对于包含mRNA的递送体系，可以使用QUANT-ITTM RNA测定(InvitrogenCorporation Carlsbad，CA)评价递送体系对RNA的包封情况。在1×TE缓冲溶液中将样品稀释至约5μg/mL的浓度。将50μL稀释过的样品转移至96孔板上并向各孔中添加50μL TE缓冲液或50μL 5％TritonX-100溶液。在37℃温度下孵育板15分钟。将试剂以1:100稀释于TE缓冲液中，并向各孔中添加100μL该溶液。可以使用荧光板读取器(Wallac Victor1420Multilabel Counter；Perkin Elmer，Waltham，MA)在例如约480nm激发波长和例如约520nm发射波长下测量荧光强度。从各样品的荧光度值中减去试剂空白的荧光度值并通过用完全样品(未添加Triton X-100)的荧光强度除以被破坏的样品(由添加Triton X-100引起)的荧光度值来测定游离RNA的百分比。其中，buffer：1×TE的荧光强度如图4所示，buffer：1×TE(5％Trion X-100)的荧光强度如图5所示；
包封率计算公式为：包封率(EE％)＝(总RNA浓度-游离mRNA浓度)/总RNA浓度，测得的本发明递送体系的包封率为94％。
实施例3
本发明实施例2制备得到的递送体系的荧光素酶mRNA体内递送性能的评价：
A.体内递送(肌肉)：每组随机选取3只小鼠，按0.25mg/kg的用量，使用肌肉注射实施例2制备的递送体系，以PBS水溶液和MC3脂质体纳米颗粒分别作为阴性和控制组对照。6小时后，分别往每只小鼠体内通过腹腔注射200μL 10mg/mL的D-荧光素钾盐，15分钟后，将小鼠放置于活体成像系统(IVIS-200，Xenogen)下，观察每只小鼠总的萤光强度，通过软件读取信号。代表性递送载体三种给药方式递送Fluc mRNA的表达强度如图6所示。代表性小鼠整体成像结果如图7所示。
由图6、图7可知：所述的递送体系与MC3控制组均在肌肉中检测到明显的阳性表达，所述递送系统的表达效率显著优于控制组对照。
B.体内递送(静脉)：每组随机选取3只小鼠，按0.75mg/kg的用量，使用尾静脉注射实施例2制备的递送体系，以PBS水溶液和MC3脂质体纳米颗粒分别作为阴性和控制组对照。6小时后，分别往每只小鼠体内通过腹腔注射200μL 10mg/mL的D-荧光素钾盐，15分钟后，将小鼠放置于活体成像系统(IVIS-200，Xenogen)下，观察每只小鼠总的萤光强度，随后在10min内解剖小鼠，取小鼠的心脏，肝脏，肾脏，肺脏，脾脏通过软件读取信号。代表性递送载体三种给药方式递送Fluc mRNA的表达强度见图8，代表性各器官成像结果如图9所示。
由图8、图9可知：所述的递送体系与MC3控制组均在肝脏中检测到明显的阳性表达，所述递送系统的表达效率显著优于控制组对照。
实施例4
本发明实施例2制备得到的递送体系的人促红细胞生成素mRNA体内递送性能的评价：
每组随机选取3只小鼠，按0.75mg/kg的用量，使用尾静脉注射实施例2制备的递送体系，以PBS水溶液和MC3脂质体纳米颗粒分别作为阴性和控制组对照。6小时后，使用二氧化碳将小鼠安乐死。通过在4℃下以5000g离心10分钟从全血中分离出血清，速冻并在80℃下储存以用于分析。根据制造商的说明，使用市售试剂盒(DEP00，R&D系统)进行ELISA分析，从测试组测得的hEPO表达水平(ng/mL)，hEPO的表达量见图10。
由图10可知：所述的递送体系与MC3控制组均在血清中检测到明显的阳性表达，所述递送系统的表达效率显著优于控制组对照。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (9)
1.一种可离子化脂质，其特征在于，所述可离子化脂质的结构式如式I所示：
2.如权利要求1所述的可离子化脂质的制备方法，其特征在于，所述制备方法包括如下步骤：
(a)将十一醇和6-溴己酸进行酯化反应，得到中间体1；
(b)将9-十七醇和8-溴辛酸进行酯化反应，得到中间体2；
(c)将中间体2和3-氨基-1，1，1-三氟丙烷-2-醇进行缩合反应，得到中间体3；
(d)将中间体3和中间体1进行缩合反应，得到所述可离子化脂质。
3.权利要求1所述的可离子化脂质在制备mRNA递送体系中的应用。
4.一种mRNA递送体系，其特征在于，包括权利要求1所述的可离子化脂质、磷脂、PEG脂质和结构性脂质和mRNA。
5.根据权利要求4所述的mRNA递送体系，其特征在于，所述可离子化脂质、结构性脂质、磷脂和PEG脂质的摩尔比为50∶(35～40)∶(9～11)∶(1.3～1.8)。
6.根据权利要求4所述的mRNA递送体系，其特征在于，所述可离子化脂质与mRNA的摩尔比为(8～15)∶1。
7.权利要求4～6任一所述的mRNA递送体系的制备方法，其特征在于，包括如下步骤：
(a)按照摩尔比，将可离子化脂质、结构性脂质、磷脂和PEG脂质溶于乙醇，得到脂质溶液；
(b)在微流控系统中，将脂质溶液以2～18mL/min的流速注入mRNA溶液中，得到纳米脂质分散液，经0.22μm无菌过滤器过滤后，得到所述mRNA递送体系。
8.根据权利要求7所述的制备方法，其特征在于，所述mRNA溶液的浓度为0.08～0.2mg/mL，溶剂为pH值为3～4、50mM的柠檬酸钠缓冲液。
9.根据权利要求7所述的制备方法，其特征在于，所述mRNA溶液和脂质溶液的体积比为(2～4)∶1。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210137654.3A CN114456081A (zh) | 2022-02-15 | 2022-02-15 | 一种可离子化脂质及其制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210137654.3A CN114456081A (zh) | 2022-02-15 | 2022-02-15 | 一种可离子化脂质及其制备方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114456081A true CN114456081A (zh) | 2022-05-10 |
Family
ID=81412928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210137654.3A Pending CN114456081A (zh) | 2022-02-15 | 2022-02-15 | 一种可离子化脂质及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114456081A (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130115274A1 (en) * | 2011-11-04 | 2013-05-09 | Nitto Denko Corporation | Method of producing lipid nanoparticles for drug delivery |
| US20170210698A1 (en) * | 2015-09-17 | 2017-07-27 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
| CN111356444A (zh) * | 2017-10-31 | 2020-06-30 | 摩登纳特斯有限公司 | 用于递送编码vegf-a多肽的经修饰的rna的脂质纳米颗粒 |
| US20210299058A1 (en) * | 2020-03-26 | 2021-09-30 | Shenzhen Neocura Biotechnology Corporation | Intracellular Delivery System for mRNA Nucleic Acid Drugs, Preparation Method and Application Thereof |
-
2022
- 2022-02-15 CN CN202210137654.3A patent/CN114456081A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130115274A1 (en) * | 2011-11-04 | 2013-05-09 | Nitto Denko Corporation | Method of producing lipid nanoparticles for drug delivery |
| US20170210698A1 (en) * | 2015-09-17 | 2017-07-27 | Modernatx, Inc. | Compounds and compositions for intracellular delivery of therapeutic agents |
| CN111356444A (zh) * | 2017-10-31 | 2020-06-30 | 摩登纳特斯有限公司 | 用于递送编码vegf-a多肽的经修饰的rna的脂质纳米颗粒 |
| US20210299058A1 (en) * | 2020-03-26 | 2021-09-30 | Shenzhen Neocura Biotechnology Corporation | Intracellular Delivery System for mRNA Nucleic Acid Drugs, Preparation Method and Application Thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022166213A1 (zh) | 一种可离子化脂质分子、其制备方法及其在制备脂质纳米颗粒中的应用 | |
| Su et al. | In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles | |
| Kim et al. | Enhanced siRNA delivery using cationic liposomes with new polyarginine-conjugated PEG-lipid | |
| US20040167090A1 (en) | Covalent modification of RNA for in vitro and in vivo delivery | |
| JP2024507482A (ja) | イオン化可能な脂質分子、その製造方法及び脂質ナノ粒子の製造における応用 | |
| WO1997000965A2 (en) | A lipidic vector for nucleic acid delivery | |
| Nishimura et al. | Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes | |
| O’Mahony et al. | In vitro investigations of the efficacy of cyclodextrin-siRNA complexes modified with lipid-PEG-Octaarginine: towards a formulation strategy for non-viral neuronal siRNA delivery | |
| Sharifnia et al. | In-vitro transcribed mRNA delivery using PLGA/PEI nanoparticles into human monocyte-derived dendritic cells | |
| Jafari et al. | A new amphipathic, amino-acid-pairing (AAP) peptide as siRNA delivery carrier: physicochemical characterization and in vitro uptake | |
| WO2023236976A1 (zh) | 一种脂质化合物及其制备方法与应用 | |
| Cohen et al. | Enhanced cell penetration of acid-degradable particles functionalized with cell-penetrating peptides | |
| KR20230042018A (ko) | 변형된 말단기를 갖는 폴리(아민-코-에스테르) 폴리머 및 향상된 폐 운반 | |
| Hyodo et al. | “Programmed packaging” for gene delivery | |
| WO2017063131A1 (zh) | 一种氧化响应去正电荷的阳离子聚合物、制备方法和应用 | |
| CA2223934A1 (en) | Separation of active complexes | |
| WO2016027699A1 (ja) | 核酸送達のためのカチオン性脂質 | |
| CN114456081A (zh) | 一种可离子化脂质及其制备方法和应用 | |
| CN115998714B (zh) | 一种脂质纳米颗粒、递送系统及递送系统的制备方法 | |
| Begum et al. | Gastrin-releasing peptide receptor-targeted hybrid peptide/phospholipid pDNA/siRNA delivery systems | |
| CN116963770B (zh) | 疫苗组合物和疫苗组合物的制备方法 | |
| CN117603078A (zh) | 新型含氟可离子化脂质、制备方法及在递送体系中的应用 | |
| CN116963770B9 (zh) | 疫苗组合物和疫苗组合物的制备方法 | |
| KR102549868B1 (ko) | 재조합 프로타민을 이용한 지질 나노입자 기반 약물 전달체 및 이의 제조방법 | |
| CN116143707B (zh) | 一种碱基可电离脂质及其制备方法与应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |