CN116509816A - 一种可电离脂质纳米颗粒及其制备方法和应用 - Google Patents
一种可电离脂质纳米颗粒及其制备方法和应用 Download PDFInfo
- Publication number
- CN116509816A CN116509816A CN202310283120.6A CN202310283120A CN116509816A CN 116509816 A CN116509816 A CN 116509816A CN 202310283120 A CN202310283120 A CN 202310283120A CN 116509816 A CN116509816 A CN 116509816A
- Authority
- CN
- China
- Prior art keywords
- chloride
- dimethylamino
- carbamate
- ethyl
- reaction
- 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 43
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical class 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 claims abstract description 26
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 51
- 238000006243 chemical reaction Methods 0.000 claims description 40
- -1 propyl (2- (dimethylamino) ethyl) Chemical group 0.000 claims description 28
- 239000012043 crude product Substances 0.000 claims description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 14
- 239000012074 organic phase Substances 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- 238000004440 column chromatography Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000003999 initiator Substances 0.000 claims description 4
- SKDHHIUENRGTHK-UHFFFAOYSA-N 4-nitrobenzoyl chloride Chemical compound [O-][N+](=O)C1=CC=C(C(Cl)=O)C=C1 SKDHHIUENRGTHK-UHFFFAOYSA-N 0.000 claims description 3
- 239000012448 Lithium borohydride Substances 0.000 claims description 3
- IPIVAXLHTVNRBS-UHFFFAOYSA-N decanoyl chloride Chemical compound CCCCCCCCCC(Cl)=O IPIVAXLHTVNRBS-UHFFFAOYSA-N 0.000 claims description 3
- NTQYXUJLILNTFH-UHFFFAOYSA-N nonanoyl chloride Chemical compound CCCCCCCCC(Cl)=O NTQYXUJLILNTFH-UHFFFAOYSA-N 0.000 claims description 3
- PQZWQGNQOVDTRF-UHFFFAOYSA-N pentadecanoyl chloride Chemical compound CCCCCCCCCCCCCCC(Cl)=O PQZWQGNQOVDTRF-UHFFFAOYSA-N 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- LPWCRLGKYWVLHQ-UHFFFAOYSA-N tetradecanoyl chloride Chemical compound CCCCCCCCCCCCCC(Cl)=O LPWCRLGKYWVLHQ-UHFFFAOYSA-N 0.000 claims description 3
- LPVCCSSTZWTHFO-UHFFFAOYSA-N 2-(dimethylamino)ethyl carbamate Chemical compound CN(C)CCOC(N)=O LPVCCSSTZWTHFO-UHFFFAOYSA-N 0.000 claims description 2
- UWPISLQLJRDZOS-UHFFFAOYSA-N Cl.Cl.CCC(=O)OC Chemical compound Cl.Cl.CCC(=O)OC UWPISLQLJRDZOS-UHFFFAOYSA-N 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims description 2
- NQGIJDNPUZEBRU-UHFFFAOYSA-N dodecanoyl chloride Chemical compound CCCCCCCCCCCC(Cl)=O NQGIJDNPUZEBRU-UHFFFAOYSA-N 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- ARBOVOVUTSQWSS-UHFFFAOYSA-N hexadecanoyl chloride Chemical compound CCCCCCCCCCCCCCCC(Cl)=O ARBOVOVUTSQWSS-UHFFFAOYSA-N 0.000 claims description 2
- FJRPWCNFWGBGOF-UHFFFAOYSA-N tridecanoyl chloride Chemical compound CCCCCCCCCCCCC(Cl)=O FJRPWCNFWGBGOF-UHFFFAOYSA-N 0.000 claims description 2
- JUKPJGZUFHCZQI-UHFFFAOYSA-N undecanoyl chloride Chemical compound CCCCCCCCCCC(Cl)=O JUKPJGZUFHCZQI-UHFFFAOYSA-N 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 17
- 108020004707 nucleic acids Proteins 0.000 abstract description 16
- 102000039446 nucleic acids Human genes 0.000 abstract description 16
- 150000007523 nucleic acids Chemical class 0.000 abstract description 16
- 229940079593 drug Drugs 0.000 abstract description 10
- 238000001890 transfection Methods 0.000 abstract description 9
- 238000005538 encapsulation Methods 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 6
- 239000002245 particle Substances 0.000 abstract description 6
- 238000012377 drug delivery Methods 0.000 abstract description 3
- 239000007787 solid Substances 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 238000004809 thin layer chromatography Methods 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- MIOQZKCEDSNQNA-UHFFFAOYSA-N methyl 2,3-diaminopropanoate;dihydrochloride Chemical compound Cl.Cl.COC(=O)C(N)CN MIOQZKCEDSNQNA-UHFFFAOYSA-N 0.000 description 8
- 229920001223 polyethylene glycol Polymers 0.000 description 8
- 239000002202 Polyethylene glycol Substances 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 230000000694 effects Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000002296 dynamic light scattering Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- XQYQGALUIGBFAM-UHFFFAOYSA-N (4-nitrophenyl) propyl carbonate Chemical compound CCCOC(=O)OC1=CC=C([N+]([O-])=O)C=C1 XQYQGALUIGBFAM-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- CLWAXFZCVYJLLM-UHFFFAOYSA-N 1-chlorohexadecane Chemical compound CCCCCCCCCCCCCCCCCl CLWAXFZCVYJLLM-UHFFFAOYSA-N 0.000 description 1
- ASZMYJSJEOGSBR-UHFFFAOYSA-N 1-chlorotridecane Chemical compound CCCCCCCCCCCCCCl ASZMYJSJEOGSBR-UHFFFAOYSA-N 0.000 description 1
- ZHKKNUKCXPWZOP-UHFFFAOYSA-N 1-chloroundecane Chemical compound CCCCCCCCCCCCl ZHKKNUKCXPWZOP-UHFFFAOYSA-N 0.000 description 1
- HVYWMOMLDIMFJA-UHFFFAOYSA-N 3-cholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 HVYWMOMLDIMFJA-UHFFFAOYSA-N 0.000 description 1
- IZVFFXVYBHFIHY-SKCNUYALSA-N 5alpha-cholest-7-en-3beta-ol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)CCCC(C)C)CC[C@H]33)C)C3=CC[C@H]21 IZVFFXVYBHFIHY-SKCNUYALSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 229930182558 Sterol Natural products 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
- 230000003698 anagen phase Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229940126672 traditional medicines Drugs 0.000 description 1
Classifications
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C201/00—Preparation of esters of nitric or nitrous acid or of compounds containing nitro or nitroso groups bound to a carbon skeleton
- C07C201/06—Preparation of nitro compounds
- C07C201/12—Preparation of nitro compounds by reactions not involving the formation of nitro groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C269/04—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups from amines with formation of carbamate groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C271/06—Esters of carbamic acids
- C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
- C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Nanotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
本发明属于生物医药领域,具体涉及一种可电离脂质纳米颗粒及其制备方法和应用。其包括可电离脂质、辅助脂质、胆固醇、PEG脂质。本发明通过对可电离脂质分子的结构进行调整,并对LNP各组分的比例进行优化筛选。以实现LNP组分方案能够充分发挥mRNA‑LNP制剂的效力并且也较好的实现靶向递送的目的,通过对本发明的可电离脂质纳米颗粒进行检测,发现粒径大多集中于80‑100nm,符合纳米药物尺度,说明所制备的LNP可顺利穿过细胞间隙,具有优异的核酸药物递送性能,包封率在60%‑95%之间,此外转染率在10%到90%之间,有良好的转染性能。
Description
技术领域
本发明属于生物医药领域,具体涉及一种可电离脂质纳米颗粒及其制备方法和应用。
背景技术
核酸药物因兼具基因修饰和传统药物的双重特点,已逐渐成为精准生物医学和疾病治疗的热点。近年来,随着对疾病基因相关机制的不断深入研究,包括siRNA、mRNA、质粒DNA及其他基因疗法在内的核酸类药物,成为当前新药开发的热门领域,其中一些核酸类药物以获得批准上市,用于临床治疗。核酸药物针对基因转录后水平治疗,候选靶点丰富,尤其一些蛋白靶点难以成药的基因,开发和应用潜力巨大。
然而,核酸类药物需要进入细胞内部才能较好地发挥其调节基因表达的作用,为实现这一目的,现有技术使用脂质纳米颗粒(LNP)来封装和递送RNA到机体特定部位。LNP主要有以下四种成分组成:1)“可离子化(或阳离子)的脂质分子”是LNP技术的关键,它的极性随着pH值的变化会发生改变,在低pH值的环境中携带正电荷,通过静电作用与电负性的核酸结合形成纳米粒,可保护核酸药物免遭核酸酶的降解,实现核酸药物在体内的稳定递送。在生理pH值时是电中性分子,减少了毒副作用。2)聚乙二醇修饰的脂质分子,聚乙二醇(PEG)的修饰具有多种功能,能够防止LNP的聚集,使LNP粒度均匀,并可降低网状内皮***的清除作用。3)胆固醇,增加LNP稳定性,促进膜融合;4)磷脂分子,为LNP双分子层提供结构,协助构成LNP的完整结构。
在体内,LNP的成分和比例发生变化会对运载效果以及mRNA在体内的生物活性产生深远影响,此外有些mRNA需要靶向递送到特定的组织才能有效发挥其作用。而现有的LNP组分方案无法充分发挥mRNA-LNP制剂的效力并且也较难实现靶向递送的目的,因此需要持续对可电离脂质分子的结构进行调整,并对LNP各组分的比例进行优化筛选。
因此,针对上述缺陷,本发明提出一种可电离脂质纳米颗粒及其制备方法和应用。
发明内容
本发明的目的是提供一种可电离脂质纳米颗粒及其制备方法和应用,解决现有的LNP组分方案无法充分发挥mRNA-LNP制剂的效力并且也较难实现靶向递送的难题。
本发明解决上述技术问题的技术方案如下:
本发明的第一个方面,提供一种可电离脂质纳米颗粒,包括下式(I)的化合物:
或其同分异构体;
其中所述R为-(CH2)nCH3。
作为本发明的一种优选技术方案,还包括辅助脂质、胆固醇、PEG脂质,其中所述辅助脂质包括DMG-PEG。
作为本发明的一种优选技术方案,所述结构(I)的化合物为:
2,3-二壬酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
2,3-二癸酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
2,3-二十一碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
2,3-二十二碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
2,3-二十三碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
2,3-二十四碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
2,3-二十五碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
2,3-二十六碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
中任一种或任一种的同分异构体。
本发明的第二个方面,提供一种上述可电离脂质纳米颗粒的制备方法,包括以下步骤:
向烧瓶中加入2,3-二氨基丙酸甲酯二盐酸盐以及N,N-二甲基甲酰胺,搅拌均匀后加入三乙胺,冰浴条件下滴加引发剂,于室温下反应2小时后,TLC检测原料反应完全,停止反应;向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品,所得粗产品通过柱层析法进行纯化分离,得到产物A;
向反应瓶中加入产物A以及四氢呋喃,于-40℃冷却10分钟后,滴加硼氢化锂溶液,反应2小时后,TLC检测原料反应完全,停止反应;向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品。所得粗产品通过柱层析法进行纯化分离,得到产物B;
向反应瓶中加入产物B、吡啶以及四氢呋喃,在冰浴中冷却10分钟后,加入4-硝基苯基羰基氯,反应2小时后,TLC检测原料反应完全,停止反应;向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得产物C;
于反应管中加入产物C、N,N-二甲基乙-1,2-二胺、三乙胺以及二氯甲烷,室温下搅拌4小时,TLC检测原料反应完全,停止反应,向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品,所得粗产品通过柱层析法进行纯化分离,得到最终产物。
作为本发明的一种优选技术方案,所述引发剂为壬酰氯、癸酰氯、十一碳酰氯、十二碳酰氯、十二碳酰氯、十三碳酰氯、十四碳酰氯、十五碳酰氯、十六碳酰氯中的任一种。
本发明的第三个方面,提供了一种可电离脂质纳米颗粒在肿瘤诊断和治疗中的应用
本发明的有益效果:
本发明通过对可电离脂质分子的结构进行调整,并对LNP各组分的比例进行优化筛选。以实现LNP组分方案能够充分发挥mRNA-LNP制剂的效力并且也较好的实现靶向递送的目的,通过对本发明的可电离脂质纳米颗粒进行检测,发现粒径大多集中于80-100nm,符合纳米药物尺度,说明所制备的LNP可顺利穿过细胞间隙,具有优异的核酸药物递送性能,包封率在60%-95%之间,此外转染率在10%到90%之间,有良好的转染性能。
附图说明
图1为本发明实施例9中动态光散射检测LNP的粒径图;
图2为本发明实施例9中动态光散射检测LNP的电位图;
图3为本发明实施例9中LNP包封率的测定结果图;
图4为实施例9中LNP转染率的测定结果图。
下面通过具体实施方式和实施例,对本发明的技术方案作进一步的详细描述。
具体实施方式
下面通过具体实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
实施例1
(1)合成2,3-二壬酰胺丙酸甲酯
向250毫升的圆底烧瓶中加入2,3-二氨基丙酸甲酯二盐酸盐(3.56mmol,0.68g)以及50毫升N,N-二甲基甲酰胺,搅拌均匀后加入三乙胺(35.56mmol,5mL)。在冰浴中向反应液滴加壬酰氯(14.22mmol,3.28mL),于室温下反应2小时后,薄层色谱法(TLC)检测原料反应完全,停止反应。向反应体系中加入水100mL,随后以二氯甲烷萃取(50mL*3),合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品。所得粗产品通过柱层析法(DCM/MeOH=30/1到10/1)进行纯化分离,得到2,3-二壬酰胺丙酸甲酯为白色固体(1.15g,81%)。1H NMR(600MHz,CDCl3)δ6.89(d,J=12Hz,1H),6.25(t,J=11.2Hz,1H),4.57(m,1H),3.72(s,3H),3.60(t,J=9.0Hz,2H),2.11-2.21(m,4H),1.58(m,4H),1.21(b,20H),0.82-0.85(t,J=11.2Hz,6H)ppm;13C NMR(150MHz,CDCl3)δ177.1,145.0,143.6,143.5 141.5,140.9,136.4,132.1,130.2(2C),128.5,127.7(2C),125.9,123.6,116.8,112.4,111.8,71.7,41.8,21.4ppm;HRMS(ESI):m/z[M+H]+calcd for C22H43N2O4 399.5960;found399.5962。
(2)合成N,N'-(3-羟基丙烷-1,2-二酰基)二壬酰胺
向25毫升反应瓶中加入2,3-二壬酰胺丙酸甲酯1-1(0.73mmol,0.35g)以及10毫升四氢呋喃,于-40℃冷却10分钟后,滴加硼氢化锂溶液(4N in THF,0.22mmol,0.055mL)。反应2小时后,TLC检测原料反应完全,停止反应。向反应体系中加入水15mL,随后以二氯甲烷萃取(20mL*3),合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品。所得粗产品通过柱层析法(DCM/MeOH=30/1到10/1)进行纯化分离,得到N,N'-(3-羟基丙烷-1,2-二酰基)二壬酰胺为白色固体(0.21g,76%)。1H NMR(600MHz,CDCl3)δ8.01(s,1H),5.52(s,1H),4.49(s,1H),3.50–3.33(m,5H),2.77(t,J=6.6Hz,2H),2.52(s,2H),2.23–2.17(m,2H),1.62–1.57(m,2H),1.39(m,2H),1.31–1.24(m,26H),0.89(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ172.6,63.7,62.7,47.4,41.3,36.5,31.9(2C),30.8,29.6,29.3(3C),28.9,28.6,27.0,25.6,22.7(2C),14.1(2C)ppm;HRMS(ESI):m/z[M+H]+calcd forC21H43N2O3 371.5860;found371.5861。
(3)合成2,3-二壬酰胺丙基(4-硝基苯基)碳酸酯
向15毫升反应瓶中加入N,N'-(3-羟基丙烷-1,2-二酰基)二壬酰胺1-2(0.57mmol,0.21g)、吡啶(0.74mmol,60μL)以及5毫升四氢呋喃,在冰浴中冷却10分钟后,加入4-硝基苯基羰基氯(0.74mmol,0.15g)。反应2小时后,TLC检测原料反应完全,停止反应。向反应体系中加入水10mL,随后以二氯甲烷萃取(15mL*3),合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品2,3-二壬酰胺丙基(4-硝基苯基)碳酸酯为浅黄色固体,该粗品直接用于下一步反应中。HRMS(ESI):m/z[M+H]+calcd for C21H43N2O3536.3336;found536.3337。
(4)合成2,3-二壬酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
于10毫升的反应管中加入2,3-二壬酰胺丙基(4-硝基苯基)碳酸酯1-3(0.08mmol,42.8mg)、N,N-二甲基乙-1,2-二胺(0.12mmol,10.5mg)、三乙胺(0.24mmol,10μL)以及1毫升二氯甲烷。将反应混合液在室温下搅拌4小时后,TLC检测原料反应完全,停止反应。向反应体系中加入水10mL,随后以二氯甲烷萃取(15ml*3),合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品。所得粗产品通过柱层析法(DCM/MeOH=30/1到10/1)进行纯化分离,得到产物2,3-二壬酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为白色固体(25mg,65%)。1H NMR(600MHz,CDCl3)δ7.17(d,J=7.8Hz,1H),6.89(t,J=6.6Hz,1H),6.03(t,J=6.6Hz,1H),4.21–4.11(m,3H),3.50–3.33(m,4H),2.77(t,J=6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H),1.31–1.24(m,20H),0.89(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ173.7,172.6,157.6,62.3,60.3,50.7,46.7(2C),41.7,38.2,36.8,36.5,31.9(2C),29.3(2C),28.9(2C),28.6(2C),25.6(2C),22.7(2C),14.1(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C28H46N3O7 536.6900;found 536.6904。
实施例2
合成2,3-二癸酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
以2,3-二氨基丙酸甲酯二盐酸盐和癸酰氯为原料,用类似于实施例1的方法可制备得到2,3-二癸酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为白色固体(51%)。1H NMR(600MHz,CDCl3)δ7.15(d,J=7.8Hz,1H),6.90(t,J=6.6Hz,1H),6.03(t,J=6.6Hz,1H),4.21–4.11(m,3H),3.61–3.29(m,4H),2.83(t,J=6.6Hz,2H),2.48(s,6H),2.21–2.15(m,4H),1.68–1.43(m,4H),1.28–1.18(m,24H),0.81(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ173.8,172.5,157.8,62.4,60.1,50.9,46.5(2C),41.5,38.2,36.8,36.5,31.8(2C),29.1(2C),28.5(2C),28.3(2C),25.5(2C),22.8(2C),14.2(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C28H57N4O4 513.7880;found 513.7881。
实施例3
合成2,3-二十一碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
以2,3-二氨基丙酸甲酯二盐酸盐和十一碳酰氯为原料,用类似于实施例1的方法可制备得到2,3-二十一碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为浅黄色固体(64%)。1HNMR(600MHz,CDCl3)δ7.23(d,J=7.8Hz,1H),6.79(t,J=6.6Hz,1H),6.10(t,J=6.6Hz,1H),4.31–4.14(m,3H),3.42–3.31(m,4H),2.61(t,J=6.6Hz,2H),2.47(s,6H),2.27–2.12(m,4H),1.64–1.52(m,4H),1.31–1.24(m,28H),0.89(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ173.2,172.6,157.3,62.3,60.5,50.7,46.9(2C),41.7,38.3,36.7,36.5,31.9(2C),29.6(4C),29.3(2C),28.9(2C),28.6(2C),25.6(2C),22.7(2C),14.3(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C30H61N4O4 541.4963;found 541.4964。
实施例4
合成2,3-二十二碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
以2,3-二氨基丙酸甲酯二盐酸盐和十二碳酰氯为原料,用类似于实施例1的方法可制备得到2,3-二十二碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为浅黄色固体(57%)。1HNMR(600MHz,CDCl3)δ7.16(d,J=7.8Hz,1H),6.84(t,J=6.6Hz,1H),6.08(t,J=6.6Hz,1H),4.35–4.09(m,3H),3.57–3.32(m,4H),2.74(t,J=6.6Hz,2H),2.57(s,6H),2.33–2.20(m,4H),1.67–1.56(m,4H),1.29–1.23(m,32H),0.88(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ173.7,172.2,157.4,62.3,60.0,50.5,46.7(2C),41.7,38.3,36.8,36.5,31.9(2C),29.4(6C),29.3(2C),28.9(2C),28.6(2C),25.6(2C),22.7(2C),14.1(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C32H65N4O4 569.5006;found 569.5007。
实施例5
合成2,3-二十三碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
以2,3-二氨基丙酸甲酯二盐酸盐和十三碳酰氯为原料,用类似于实施例1的方法可制备得到2,3-二十三碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为白色固体(65%)。1HNMR(600MHz,CDCl3)δ7.17(d,J=7.8Hz,1H),6.89(t,J=6.6Hz,1H),6.03(t,J=6.6Hz,1H),4.21–4.11(m,3H),3.50–3.33(m,4H),2.77(t,J=6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H),1.31–1.24(m,36H),0.89(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ173.8,172.6,157.8,62.3,60.3,50.7,46.7(2C),41.7,38.3,36.8,36.5,31.9(2C),29.6(8C),29.3(2C),28.9(2C),28.6(2C),25.6(2C),22.7(2C),14.1(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C34H69N4O4 597.5319;found 597.5319。
实施例6
2,3-二十四碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
以2,3-二氨基丙酸甲酯二盐酸盐和十四碳酰氯为原料,用类似于实施例1的方法可制备得到2,3-二十四碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为浅黄色固体(61%)。1HNMR(600MHz,CDCl3)δ7.17(d,J=7.8Hz,1H),6.89(t,J=6.6Hz,1H),6.03(t,J=6.6Hz,1H),4.21–4.11(m,3H),3.50–3.33(m,4H),2.77(t,J=6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H),1.31–1.24(m,40H),0.89(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ175.1,174.4,156.7,63.6,57.9,49.3,44.5(2C),41.0,37.5,36.7(2C),31.9(2C),29.8(4C),29.7(4C),29.6(2C),29.5(2C),29.4(2C),29.3(2C),25.8,25.7,22.7(2C),14.1(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C36H73N4O4 625.5632;found 625.5635。
实施例7
2,3-二十五碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
以2,3-二氨基丙酸甲酯二盐酸盐和十五碳酰氯为原料,用类似于实施例1的方法可制备得到2,3-二十五碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为白色固体(53%)。1HNMR(600MHz,CDCl3)δ7.14(d,J=7.8Hz,1H),6.79(t,J=6.6Hz,1H),6.10(t,J=6.6Hz,1H),4.27–4.10(m,3H),3.52–3.31(m,4H),2.79(t,J=6.6Hz,2H),2.54(s,6H),2.25–2.15(m,4H),1.68–1.58(m,4H),1.31–1.25(m,44H),0.84(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ175.5,174.4,156.7,63.6,57.9,49.3,44.5(2C),41.0,37.5,36.7(2C),31.9(2C),29.8(4C),29.7(4C),29.6(4C),29.5(2C),29.4(2C),29.3(2C),25.8,25.7,22.7(2C),14.1(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C38H77N4O4 653.5945;found 653.5947。
实施例8
2,3-二十六酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯
以2,3-二氨基丙酸甲酯二盐酸盐和十六碳酰氯为原料,用类似于实施例1的方法可制备得到2,3-二十六酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯为白色固体(64%)。1HNMR(600MHz,CDCl3)δ7.17(d,J=7.8Hz,1H),6.89(t,J=6.6Hz,1H),6.03(t,J=6.6Hz,1H),4.21–4.11(m,3H),3.50–3.33(m,4H),2.77(t,J=6.6Hz,2H),2.52(s,6H),2.23–2.17(m,4H),1.62–1.57(m,4H),1.31–1.24(m,48H),0.89(t,J=7.8Hz,6H)ppm;13C NMR(125MHz,CDCl3)δ175.0,174.3,156.8,63.5,58.0,50.1,44.9(2C),41.1,38.1,36.8,36.7(4C),31.9(4C),29.7(4C),29.7(2C),29.6(2C),29.4(2C),29.4,29.3(2C),29.3,25.8,25.7,22.7(2C),14.1(2C)ppm;HRMS(ESI):m/z[M+H]+calcd for C40H81N4O4 681.6258;found681.6259。
实施例9
以上所述的本发明实施例设计合成的可电离脂质分子主要用于制备LNP,具体操作如下:
将可电离脂质、胆固醇、PEG、辅助脂质溶于乙醇中,将编码表达EGFP的mRNA溶于pH=4.0的柠檬酸盐缓冲液中。将混合脂质溶液和mRNA溶液按照表1中的处方用微流控法混合得到mRNA-LNP。
通过微流控法合成LNP
借助微流控合成仪(NanoAssemblr)及随厂的微流控芯片。预处理微流控芯片通道后,一侧的注射器吸入900μL的mRNA缓冲液,另外一侧的注射器吸入450μL的含有各组分的乙醇溶液。具体参数如下:总流速为12ml/min,水相/有机相为2:1,收集所有的产物LNP溶液,随后加入1×PBS缓冲液稀释至溶液中乙醇比例小于0.5%。
表1中,mRNA浓度为1mg/mL,体积为40μL且水相总体积为900μL,可电离脂质浓度为10mg/mL,体积为40μL且有机相总体积为450μL,胆固醇浓度为5mg/mL,PEG浓度为5mg/mL,辅助脂质浓度为5mg/mL,可电离脂质与mRNA的重量比约为10:1,混合方法为微流控法。通过形成的8组样品,进行以下的测试。
样品 | 可电离脂质 | 胆固醇 | PEG | 辅助脂质 | 固醇(μL) | PEG(μL) | DOPE(μL) | 乙醇(μL) |
1 | 1 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
2 | 2 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
3 | 3 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
4 | 4 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
5 | 5 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
6 | 6 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
7 | 7 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
8 | 8 | 胆固醇 | C14-PEG2K | DOPE | 50.5 | 8.2 | 48.5 | 152.8 |
表1
为了测量LNP的尺度,进行了mRNA-LNP的粒径与电位表征:
使用Malvern Zetasizer Nano ZS,以90°反向散射检测模式,利用动态光散射检测LNP的粒径和电位。结果如图1、图2所示。图1所示LNP粒径范围在70-110nm,大多集中于80-100nm,符合纳米药物尺度,说明所制备的LNP可顺利穿过细胞间隙,具有优异的核酸药物递送性能。图2所示ζ电位在-6~6mV之间,证明LNP制剂体系稳定,不易聚集或沉降。
为了测量LNP的包封率,进行mRNA-LNP的包封率表征:
使用Quant-iTTM RNA试剂及多模式微孔板检测***Mutimode PlateReader(EnSight),测定LNP包封率。Quant-iTTM/>RNA试剂是无法透过LNP,因此只有游离的未被LNP包载的核酸可以被结合。Triton-100作为一种表面活性剂常被用做破乳剂,使用2%的Triton-100处理获得的LNP可以使包载的核酸释放,得到总核酸量。通过计算破乳前后核酸量的差异得到载药量,再除以总核酸量即可得到包封率。测得本系列产品包封率在60%-95%之间,结果如附图3所示。
为了测量LNP的转染率,进行mRNA-LNP的转染效率表征:
取对数生长期的Hep3B细胞接种到6孔细胞板(20万细胞/孔)培养过夜,待细胞密度达到80%以上,弃掉培养基并使用1X PBS洗涤3次,取1mL无血清DMEM培养基配制的EGFPmRNA-LNP(1μg/mL)溶液,加入到细胞孔中并设置3个复孔。6小时后弃掉培养基,更换为有血清的正常DMEM培养基继续培养24小时,使用流式细胞仪检测细胞的荧光比例。结果如附图4所示,转染率在10%到90%之间,有良好的转染性能。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种可电离脂质纳米颗粒,其特征在于,包括下式(I)的化合物:
或其同分异构体;
其中所述R为-(CH2)nCH3。
2.根据权利要求1所述的可电离脂质纳米颗粒,其特征在于,还包括辅助脂质、胆固醇、PEG脂质,其中所述辅助脂质包括DMG-PEG。
3.根据权利要求1所述的可电离脂质纳米颗粒,其特征在于,所述结构(I)的化合物为:
2,3-二壬酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯、
2,3-二癸酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯、
2,3-二十一碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯、
2,3-二十二碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯、
2,3-二十三碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯、
2,3-二十四碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯、
2,3-二十五碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯、
2,3-二十六碳酰胺丙基(2-(二甲氨基)乙基)氨基甲酸酯的任一种或任一种的同分异构体。
4.一种制备权利要求1~3任一项所述的可电离脂质纳米颗粒的方法,其特征在于,具体的制备步骤包括:
向烧瓶中加入2,3-二氨基丙酸甲酯二盐酸盐以及N,N-二甲基甲酰胺,搅拌均匀后加入三乙胺,冰浴条件下滴加引发剂,于室温下反应2小时后,TLC检测原料反应完全,停止反应;向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品,所得粗产品通过柱层析法进行纯化分离,得到产物A;
向反应瓶中加入产物A以及四氢呋喃,于-40℃冷却10分钟后,滴加硼氢化锂溶液,反应2小时后,TLC检测原料反应完全,停止反应;向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品。所得粗产品通过柱层析法进行纯化分离,得到产物B;
向反应瓶中加入产物B、吡啶以及四氢呋喃,在冰浴中冷却10分钟后,加入4-硝基苯基羰基氯,反应2小时后,TLC检测原料反应完全,停止反应;向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得产物C;
于反应管中加入产物C、N,N-二甲基乙-1,2-二胺、三乙胺以及二氯甲烷,室温下搅拌4小时,TLC检测原料反应完全,停止反应,向反应体系中加入水,随后以二氯甲烷萃取,合并有机相,饱和氯化钠洗、无水硫酸钠干燥,过滤后真空浓缩得粗产品,所得粗产品通过柱层析法进行纯化分离,得到最终产物。
5.根据权利要求4所述的可电离脂质纳米颗粒的制备方法,其特征在于,所述引发剂为壬酰氯、癸酰氯、十一碳酰氯、十二碳酰氯、十二碳酰氯、十三碳酰氯、十四碳酰氯、十五碳酰氯、十六碳酰氯中的任一种。
6.一种如权利要求1~3任一项所述的可电离脂质纳米颗粒在肿瘤诊断和治疗中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310283120.6A CN116509816A (zh) | 2023-03-22 | 2023-03-22 | 一种可电离脂质纳米颗粒及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310283120.6A CN116509816A (zh) | 2023-03-22 | 2023-03-22 | 一种可电离脂质纳米颗粒及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116509816A true CN116509816A (zh) | 2023-08-01 |
Family
ID=87392947
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310283120.6A Pending CN116509816A (zh) | 2023-03-22 | 2023-03-22 | 一种可电离脂质纳米颗粒及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116509816A (zh) |
-
2023
- 2023-03-22 CN CN202310283120.6A patent/CN116509816A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111902397B (zh) | 改进了细胞内动力学的阳离子脂质 | |
EP4282855A1 (en) | Ionizable lipid molecule, preparation method therefor, and application thereof in preparation of lipid nanoparticle | |
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 | |
EP3640237B1 (en) | Lipid membrane structure for delivery into sirna cell | |
EP3842412B1 (en) | Biodegradable lipids for delivery of active compounds into tumor cells comprising two or more tertiary amino groups | |
CA3215389C (en) | Ionizable lipids and compositions for nucleic acid delivery | |
EP2319519B1 (en) | Composition for inhibiting expression of target gene | |
CN104352440A (zh) | 一种阳离子脂质体核酸类药物制剂及其制备方法和应用 | |
CN113372226B (zh) | 一种脂质分子、脂质纳米粒及其制备方法和应用 | |
CN113121381B (zh) | 一种神经酰胺类化合物及其阳离子脂质体、制备方法和应用 | |
CN116199646B (zh) | 一种基于Tris的可电离脂质及其制备方法与应用 | |
CN117623978A (zh) | 可生物降解的氨基酸衍生可电离脂质及其制备方法与应用 | |
CN116509816A (zh) | 一种可电离脂质纳米颗粒及其制备方法和应用 | |
CN116082275A (zh) | 一种脾脏中高表达的阳离子脂质化合物、包含其的组合物及用途 | |
CN116143707B (zh) | 一种碱基可电离脂质及其制备方法与应用 | |
CN106188230A (zh) | 一种阳离子脂质类化合物及其制备方法与应用 | |
CN116535381B (zh) | 具有五元环缩醛结构的阳离子脂质化合物、包含其的组合物及用途 | |
RU2747559C1 (ru) | Незаряженный липид, композиция на его основе с поликатионным амфифилом и нейтральным фосфолипидом и способ ее получения для доставки нуклеиновых кислот in vitro | |
CN117257965B (zh) | 一种核酸递送载体组合物及其应用 | |
CN114436994B (zh) | 金刚烷尾链脂质及其在细胞转染中的应用 | |
CN114315606B (zh) | 一种脂样分子及其应用 | |
CN117229160B (zh) | 三酯类阳离子脂质化合物、包含其的组合物及用途 | |
TW201815736A (zh) | 作為陽離子性脂質之化合物 | |
CN118497281A (zh) | 一种高效低毒细胞转染试剂及其应用 | |
CN117843603A (zh) | 一种可电离脂质材料及其在制备核酸递送载体中的应用 |
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 |