CN117126187A - Cationic lipid, preparation method thereof, nanoparticle assembly and application - Google Patents
Cationic lipid, preparation method thereof, nanoparticle assembly and application Download PDFInfo
- Publication number
- CN117126187A CN117126187A CN202311048251.2A CN202311048251A CN117126187A CN 117126187 A CN117126187 A CN 117126187A CN 202311048251 A CN202311048251 A CN 202311048251A CN 117126187 A CN117126187 A CN 117126187A
- Authority
- CN
- China
- Prior art keywords
- formula
- cationic lipid
- equal
- nucleic acid
- compound
- 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
- -1 Cationic lipid Chemical class 0.000 title claims abstract description 71
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title description 9
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 33
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 33
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 33
- 239000000126 substance Substances 0.000 claims abstract description 33
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 18
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 18
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 15
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 11
- 238000001338 self-assembly Methods 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims description 114
- 239000003054 catalyst Substances 0.000 claims description 14
- 229910052739 hydrogen Inorganic materials 0.000 claims description 14
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 claims description 12
- ISAOCJYIOMOJEB-UHFFFAOYSA-N benzoin Chemical compound C=1C=CC=CC=1C(O)C(=O)C1=CC=CC=C1 ISAOCJYIOMOJEB-UHFFFAOYSA-N 0.000 claims description 12
- 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 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 239000002243 precursor Substances 0.000 claims description 9
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 8
- 238000007259 addition reaction Methods 0.000 claims description 7
- 125000005843 halogen group Chemical group 0.000 claims description 7
- 244000028419 Styrax benzoin Species 0.000 claims description 6
- 235000000126 Styrax benzoin Nutrition 0.000 claims description 6
- 235000008411 Sumatra benzointree Nutrition 0.000 claims description 6
- 229960002130 benzoin Drugs 0.000 claims description 6
- 235000012000 cholesterol Nutrition 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 6
- 235000019382 gum benzoic Nutrition 0.000 claims description 6
- 150000002632 lipids Chemical class 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 125000002572 propoxy group Chemical group [*]OC([H])([H])C(C([H])([H])[H])([H])[H] 0.000 claims description 3
- 125000001494 2-propynyl group Chemical group [H]C#CC([H])([H])* 0.000 claims description 2
- 125000002947 alkylene group Chemical group 0.000 claims description 2
- 150000001768 cations Chemical class 0.000 claims description 2
- 125000000753 cycloalkyl group Chemical group 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 125000001302 tertiary amino group Chemical group 0.000 claims 3
- 239000003814 drug Substances 0.000 claims 1
- 229920006395 saturated elastomer Polymers 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 125000002091 cationic group Chemical group 0.000 abstract description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 7
- 239000001301 oxygen Substances 0.000 abstract description 7
- 229910052760 oxygen Inorganic materials 0.000 abstract description 7
- 230000004044 response Effects 0.000 abstract description 7
- 238000005538 encapsulation Methods 0.000 abstract description 5
- 230000001105 regulatory effect Effects 0.000 abstract description 4
- 230000008859 change Effects 0.000 abstract description 3
- 230000001276 controlling effect Effects 0.000 abstract description 3
- 238000011068 loading method Methods 0.000 abstract description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 30
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 27
- 239000002904 solvent Substances 0.000 description 25
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 24
- 238000006243 chemical reaction Methods 0.000 description 24
- 238000001819 mass spectrum Methods 0.000 description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 18
- 239000003208 petroleum Substances 0.000 description 18
- 238000000746 purification Methods 0.000 description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 14
- 239000000463 material Substances 0.000 description 14
- 108700021021 mRNA Vaccine Proteins 0.000 description 13
- 229940126582 mRNA vaccine Drugs 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000001257 hydrogen Substances 0.000 description 12
- 238000004949 mass spectrometry Methods 0.000 description 12
- 238000001228 spectrum Methods 0.000 description 12
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 12
- 238000004440 column chromatography Methods 0.000 description 11
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 11
- UKLDJPRMSDWDSL-UHFFFAOYSA-L [dibutyl(dodecanoyloxy)stannyl] dodecanoate Chemical compound CCCCCCCCCCCC(=O)O[Sn](CCCC)(CCCC)OC(=O)CCCCCCCCCCC UKLDJPRMSDWDSL-UHFFFAOYSA-L 0.000 description 10
- 239000003480 eluent Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 108020004999 messenger RNA Proteins 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- 150000003512 tertiary amines Chemical group 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 8
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 8
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Substances OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- 229960005486 vaccine Drugs 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 239000002502 liposome Substances 0.000 description 5
- PSGAAPLEWMOORI-PEINSRQWSA-N medroxyprogesterone acetate Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2CC[C@]2(C)[C@@](OC(C)=O)(C(C)=O)CC[C@H]21 PSGAAPLEWMOORI-PEINSRQWSA-N 0.000 description 5
- 239000012299 nitrogen atmosphere Substances 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 230000004043 responsiveness Effects 0.000 description 5
- 238000000429 assembly Methods 0.000 description 4
- 230000000712 assembly Effects 0.000 description 4
- 239000004305 biphenyl Substances 0.000 description 4
- 235000010290 biphenyl Nutrition 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 4
- 229910000027 potassium carbonate Inorganic materials 0.000 description 4
- 238000012827 research and development Methods 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 238000003848 UV Light-Curing Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- WWYFPDXEIFBNKE-UHFFFAOYSA-N 4-(hydroxymethyl)benzoic acid Chemical compound OCC1=CC=C(C(O)=O)C=C1 WWYFPDXEIFBNKE-UHFFFAOYSA-N 0.000 description 1
- 101100341230 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) isn-1 gene Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000006392 deoxygenation reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 150000002433 hydrophilic molecules Chemical class 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 150000002513 isocyanates Chemical class 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical compound OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003981 vehicle Substances 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
- C07F5/02—Boron compounds
Abstract
The invention discloses a cationic lipid, the structure of which is shown as a formula (I) or a formula (II):
Description
Technical Field
At least one embodiment of the invention relates to a cationic lipid, in particular to a cationic lipid capable of triggering dissociation, a preparation method, a nanoparticle assembly and application thereof.
Background
mRNA vaccines have been attracting attention as a novel vaccine technology. The development and use of mRNA vaccines has many advantages, including short production cycle, high flexibility, no need for living pathogens, ability to customize against multiple pathogens and variants, and the like.
However, delivery of mRNA vaccines remains a challenge. mRNA molecules have poor stability in vivo and are susceptible to enzymatic hydrolysis and immune reactions, and thus delivery materials are needed to ensure vaccine effectiveness and stability. The delivery material can help the mRNA molecules to be better stabilized and protected in vivo, while also enhancing the immunogenicity and antigen specificity of the vaccine.
Disclosure of Invention
In view of the above, the present invention provides a cationic lipid, a preparation method thereof, a nanoparticle assembly and an application thereof, so as to improve the delivery efficiency of nucleic acid substances such as mRNA and rapidly release the loaded nucleic acid substances such as mRNA.
The invention provides a cationic lipid, the structure of which is shown as a formula (I) or a formula (II):
wherein n is more than or equal to 0 and less than or equal to 10;
hydrophilic polyethylene glycol blocks having a single molecular weight;
is composed of one or two C 2 ～C 21 A hydrophobic block of alkyl chains;
R 1 ～R 7 are each independently selected from-H, halogen atom, -CH 3 、-OCH 3 、
R of at least one of the n repeating units 1 、R 2 、R 3 And R is 4 At least one of the choices
R has a tertiary amine group and is bonded to the propoxy group by a thioether bond to form a group
According to the cationic lipid provided by the embodiment of the invention, the cationic lipid comprises the hydrophilic polyethylene glycol block with single molecular weight, the hydrophobic block formed by the alkyl chain and the cationic group positioned between the hydrophilic polyethylene glycol block and the hydrophobic block, the self-assembly of the cationic lipid is realized based on the structures of the hydrophilic polyethylene glycol block and the hydrophobic block, the cationic group R can be combined with nucleic acid substances, and the cationic lipid has the capability of accurately regulating and controlling the nucleic acid loading capacity by regulating and controlling the number of the cationic groups in a proper range, so that the encapsulation efficiency of the nucleic acid substances of the cationic lipid can be increased, and the delivery efficiency of the nucleic acid substances is improved.
According to the cationic lipid provided by the embodiment of the invention, the nanoparticle assembly formed by the cationic lipid loads nucleic acid substances and delivers the nucleic acid substances into cells. The nanoparticle assembly undergoes a physical response based on an acid-base environmental change and a chemical response based on active oxygen within the cell. Based on the dual responsiveness of the nanoparticle assembly, the cargo can be released rapidly.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of a compound represented by formula C4 prepared according to example 1 of the present invention;
FIG. 2 is a mass spectrum of a compound represented by formula C4 prepared according to example 1 of the present invention;
FIG. 3 is a nuclear magnetic hydrogen spectrum of a compound of formula C5 prepared according to example 1 of the present invention;
FIG. 4 is a mass spectrum of a compound represented by formula C5 prepared according to example 1 of the present invention;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by formula C7 prepared according to example 1 of the present invention;
FIG. 6 is a mass spectrum of a compound represented by formula C7 prepared according to example 1 of the present invention;
FIG. 7 is a mass spectrum of a compound represented by formula C11 prepared according to example 1 of the present invention;
FIG. 8 is a mass spectrum of a compound represented by formula C12 prepared according to example 1 of the present invention;
FIGS. 9A-9B are SEM images of nanoparticle assemblies prepared according to example 1 of the invention;
FIG. 10 is a mass spectrum of a compound represented by formula C13 prepared according to example 2 of the present invention;
FIG. 11 is a mass spectrum of a compound represented by formula C15 prepared according to example 2 of the present invention;
FIG. 12 is a mass spectrum of a compound represented by formula C17 prepared according to example 2 of the present invention;
FIG. 13 is a mass spectrum of a compound represented by formula C20 prepared according to example 2 of the present invention;
FIG. 14 is a mass spectrum of a compound represented by formula C22 prepared according to example 3 of the present invention; and
fig. 15A to 15D are characterization results of the comparative test for responsiveness according to example 3 of the present invention.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, the size of layers and regions, as well as the relative sizes, may be exaggerated for the same elements throughout.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The terms "comprises," "comprising," and/or the like, as used herein, specify the presence of stated features, steps, operations, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, or components.
In recent years, research and development of mRNA vaccine delivery materials have made important progress, and more choices are provided for research and development and application of mRNA vaccines. The current delivery material systems are mainly of the following three types: 1) Liposome delivery systems, which are microvesicles composed of phospholipids and cholesterol, encapsulate mRNA molecules and exert a delivery effect in vivo. The liposome delivery system has the advantages of high delivery efficiency, good stability, strong biocompatibility and the like, and has become one of the main modes of mRNA vaccine delivery. For example, liposome delivery systems are used to prepare mRNA vaccines. 2) The polymer delivery system is a high molecular material formed by connecting a plurality of monomer molecules through chemical bonds, and has the characteristics of adjustable structure, good biocompatibility and the like. A polymer delivery system is a method of combining mRNA molecules with a polymeric material for delivery by forming nanoparticles. Polymer delivery systems have higher stability and delivery efficiency than liposome delivery systems, but also have certain toxicity and immunogenicity issues. In recent years, polymer delivery systems have been widely used in the fields of mRNA vaccines, gene therapy, and the like. 3) Other delivery systems in addition to liposome and polymer delivery systems, there are some other delivery systems used for the development of mRNA vaccines. For example, some researchers use nanoparticles or magnetic particles as delivery vehicles to improve the delivery efficiency and stability of mRNA molecules. In addition, some new delivery systems are also under investigation and development, such as hydrogels, gold nanoparticles, and the like. In general, research and development of mRNA vaccine delivery materials has progressed significantly in recent years, and new delivery systems and materials continue to emerge. These delivery systems play an important role in improving the delivery efficiency, stability and safety of mRNA vaccines. However, these materials have problems such as side effects, generation of immune response, and the like, and further improvement and optimization are required. Therefore, there is significant urgency in developing safer and more effective mRNA vaccine delivery materials. On the one hand, this helps to increase the delivery efficiency and stability of the mRNA vaccine, leading to better protection and immunogenicity of the vaccine in vivo. On the other hand, the method can promote research and development of vaccines, accelerate marketing and application of the vaccines, and has important significance for development of global public health industry.
In view of the above, the present invention provides a cationic lipid capable of triggering dissociation to improve the delivery efficiency of nucleic acid substances and the release rate of nucleic acid substances.
According to an exemplary embodiment of the present invention, there is provided a cationic lipid having a structure represented by formula (I) or formula (II):
wherein n is more than or equal to 1 and less than or equal to 10;
a hydrophilic polyethylene glycol block having a single molecular weight;
is composed of one or two C 2 ～C 21 A hydrophobic block of alkyl chains;
R 1 ～R 7 are each independently selected from-H, halogen atom, -CH 3 、-OCH 3 、
R of at least one of the n repeating units 1 、R 2 、R 3 And R is 4 At least one of the choices
R has a tertiary amine group and is bonded to the propoxy group by a thioether bond to form a group
According to an embodiment of the invention, R 1 -R 4 Preferably is-H or-OCH 3 ；R 5 -R 7 preferably-H or-OCH 3 。
According to the embodiment of the invention, the cationic lipid is formed by adopting the hydrophilic polyethylene glycol block with single molecular weight, so that the cationic lipid is more stable, and the stronger regulation and control capability on the cationic lipid is realized.
According to the embodiment of the invention, the cationic lipid comprises a hydrophilic polyethylene glycol block with single molecular weight, a hydrophobic block formed by alkyl chains and a cationic group positioned between the hydrophilic polyethylene glycol block and the hydrophobic block, wherein the hydrophilic polyethylene glycol block and the hydrophobic block are used for realizing self-assembly of nano particles, and the cationic group R can be combined with nucleic acid substances.
In accordance with an embodiment of the present invention,selected from any one of structures shown in formulas (A1) to (A5):
wherein a is more than or equal to 1 and less than or equal to 18; r is R 8 、R 9 Are independently selected from-H, -OCH 3 、C 1 ～C 20 Alkyl, C of (2) 3 ～C 20 Cycloalkyl, C 2 ～C 20 Alkylene or C of (2) 2 ～C 20 Is a propargyl group of (c).
In accordance with an embodiment of the present invention,preferably A1 or A3.
In accordance with an embodiment of the present invention,selected from any one of structures shown in formulas (P1) to (P9):
wherein b is more than or equal to 5 and less than or equal to 45,5, m is more than or equal to 45; r is R 10 、R 11 Are each independently selected from-H, halogen atom, -CH 3 。
According to an embodiment of the invention, 1.ltoreq.m.ltoreq.18.
In accordance with an embodiment of the present invention,preferably P1, P4, P5 or P9;
according to an embodiment of the invention, a single molecular weight polyethylene glycol linear chain is prepared by the following route:
wherein R is 12 Is C 1 ～C 100 Alkyl, C of (2) 1 ～C 100 G is more than or equal to 0 and less than or equal to 5, and k is more than or equal to 0 and less than or equal to 5.
According to an embodiment of the present invention, X is selected from any one of the structures shown below:
according to an embodiment of the present invention, R includes any one of structures shown as formulas (N1) to (N6):
wherein c is more than or equal to 0 and less than or equal to 14, d is more than or equal to 0 and less than or equal to 16,0, e is more than or equal to 7, and f is more than or equal to 0 and less than or equal to 17.
According to an embodiment of the invention, c is 1 or 2, d is 1 or 2, e is 1 or 2.
According to an embodiment of the invention, R is preferably N1 or N2.
According to the embodiment of the invention, the cationic group R has a tertiary amine structure and can be combined with nucleic acid analogues such as DNA, RNA and the like, so that the loading capacity of the nucleic acid substances is improved in the self-assembly process.
According to an exemplary embodiment of the present invention, there is provided a method for preparing a cationic lipid, comprising: and (3) carrying out addition reaction on the cationic lipid precursor and a compound RH with a sulfhydryl terminal and a tertiary amine group to obtain the cationic lipid with the structure shown in the formula (I) or the formula (II).
According to an embodiment of the present invention, the cationic lipid precursor has a structure as shown in formula (I-1) or formula (II-1):
wherein n is more than or equal to 1 and less than or equal to 10;
a hydrophilic polyethylene glycol block having a single molecular weight;
is composed of one or two C 2 ～C 21 A hydrophobic block of alkyl chains;
r1 'to R7' are each independently selected from the group consisting of-H, halogen atom, -CH 3 、-OCH 3 Or (b)
R of at least one of the n repeating units 1 ’、R 2 ’、R 3 ' and R 4 At least one of the choices
R has a tertiary amine group and forms a sulfhydryl terminus with H.
According to an embodiment of the present invention, the conditions of the addition reaction include: the addition reaction is carried out under the action of a catalyst and under the irradiation of ultraviolet light and at a heating temperature of 25-45 ℃.
According to the embodiment of the invention, in the addition reaction, the catalyst used can be benzoin dimethyl ether; the irradiation time of the ultraviolet light is 2 to 8 hours, for example, 2 hours, 4 hours, 5 hours, 6 hours and 8 hours.
According to embodiments of the invention, the catalyst benzoin dimethyl ether is used in an amount of 0.1 equivalents to 1.0 equivalents.
According to embodiments of the present invention, the reaction temperature is 25℃to 45℃and may be, for example, 25℃30℃35℃40℃45 ℃.
According to an embodiment of the invention, the hydrophilic material is selected fromGradually accessing degradable building elements from the beginning, and then utilizingEnd-capping to obtain a cationic lipid precursor shown as a formula (I-1) or a formula (II-1); then, the compound RH with the sulfhydryl end and the tertiary amine is reacted with a cationic lipid precursor shown as a formula (I-1) or a formula (II-1) to obtain the cationic lipid capable of triggering dissociation with the structure shown as the formula (I) or the formula (II). The cationic lipid prepared by the method can trigger degradation under the stimulation of active oxygen (similar to domino). In the preparation process of the cationic lipid, any toxic and expensive catalyst is not required, and meanwhile, each step of reaction is very efficient, so that the preparation cost is reduced.
The number of the degradable building elements which are gradually connected can be adjusted according to the number of nucleic acid substances which are required to be loaded.
According to an exemplary embodiment of the present invention, the present invention also provides a nanoparticle assembly including: the cationic lipid described above; and at least one nucleic acid substance, wherein each nucleic acid substance is combined with the cationic lipid through an R group, and a nanoparticle assembly is formed through self-assembly.
The invention also provides a preparation method of the nanoparticle assembly, which comprises the following steps: and combining the cationic lipid, PEG-lipid, cholesterol and nucleic acid substances by utilizing a microfluidic or nano flash precipitation technology, and obtaining the nanoparticle assembly through self-assembly.
According to an embodiment of the present invention, the nanoparticle assembly is loaded with a nucleic acid substance, and the nucleic acid substance is delivered into a cell. The nanoparticle assemblies undergo both physical and chemical responses within the cell. Based on the dual responsiveness of the nanoparticle assembly, the cargo can be released rapidly. Specifically, after the nanoparticle assembly enters cells, the acting force of N groups on R groups of cationic lipid and mRNA charges is weakened after the acid-base environment is changed, and physical response is generated in the cells by the nanoparticle assembly, so that nucleic acid substances such as mRNA and the like are released.
According to the embodiment of the invention, the nanoparticle assembly has responsiveness to active oxygen in cells, and chemical response occurs in the cells, so that the cationic lipid wrapping the nucleic acid substance is broken, and the release of the nucleic acid substance is accelerated. Specifically, under the action of intracellular active oxygen, phenylboronate is oxidized and removed, and complete degradation of a main chain is further realized through one 1, 4-elimination electron rearrangement and n1, 6-elimination, so that the disintegration of the nanoparticle assembly is facilitated and the release of carrier nucleic acid substances is promoted. The release of the nucleic acid from the nanoparticle assembly is as follows:
the method of preparing the cationic lipid and nanoparticle assembly including the cationic lipid is schematically described below. It should be noted that the examples are only specific embodiments of the present invention and are not intended to limit the scope of the present invention.
Example 1
The raw materials used in the examples are described below: benzoin dimethyl ether (DMPA), diphenyl azide phosphate (DPPA), triethylamine (TEA) and N-methylpyrrolidone (NMP) were purchased from Sigma-Aldrich company and used without further purification. Tetrahydrofuran was dried over sodium wire and refluxed, and distilled for use. Other reagents such as petroleum ether, ethyl acetate, dibutyl tin laurate, potassium carbonate, methanol and the like are all analytically pure and are purchased from national pharmaceutical chemicals company, inc., and are used without further purification. The ultrapure water used in the experiment is prepared by a Mi1li-QSP intelligent ultrapure water system, and the resistivity is 18.4MΩ & cm.
In the present embodiment, n is 2,a1, a is 1, R 1 is-H, R 2 Is->R 3 ～R 7 is-H->P1, b is 8, X is-H, R is N1, c is 1, and d is 1.
(1) Preparation of hydrophilicMolecules:
the compound (2.00 g,2.61 mmol) represented by the formula C1, the compound (0.4 g,2.61 mmol) represented by the formula C2 and potassium carbonate (0.72 g,5.22 mmol) were dissolved in 30mL of Dimethylformamide (DMF), reacted under stirring at 80℃under nitrogen atmosphere for 8 to 10 hours, the solvent was removed by rotary evaporation under reduced pressure after the completion of the reaction, dissolved in 50mL of ethyl acetate, washed twice with saturated brine, dried by adding anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. Petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain a corresponding product, which is marked as a compound shown as a formula C3, and the yield is 80-85%.
The compound (1.00 g,1.34 mmol) shown in formula C3 and potassium hydroxide (0.23 g,4.02 mmol) are dissolved in 20mL of methanol, the reaction is stirred under reflux for 10-12 h, 50mL of water is added to the solvent after the reaction is completed, the pH is adjusted to 3 by using hydrochloric acid, then ethyl acetate is added to the system, extraction is carried out, the organic phase is washed twice by saturated saline, anhydrous sodium sulfate is added for drying, the solvent is removed by rotary evaporation under reduced pressure, and the corresponding product is obtained and is recorded as the compound shown in formula C4, and the yield is 90-99%.
The compound shown in the formula C4 is characterized by nuclear magnetic hydrogen spectrum and mass spectrum, and the results are shown in figures 1 and 2.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by formula C4 prepared according to example 1 of the present invention.
FIG. 2 is a mass spectrum of a compound represented by formula C4 prepared according to example 1 of the present invention.
Referring to fig. 1 and 2, nuclear magnetic hydrogen spectrum and mass spectrum test results show that the compound shown as the formula C4 is prepared. Wherein the abscissa of fig. 2 represents the molecular weight.
Dissolving a compound (1.00 g,1.36 mmol) shown in a formula C4 and triethylamine (TEA, 0.14g,1.36 mmol) in 20mL of Tetrahydrofuran (THF), dropwise adding diphenyl azide phosphate (DPPA, 0.37g,1.36 mmol) at room temperature, stirring at room temperature for reaction for 2-18 h, removing a solvent after the reaction is finished, and separating and purifying by using petroleum ether-ethyl acetate as a eluting agent column chromatography to obtain a corresponding product, wherein the compound is shown in a formula C5, the compound is a hydrophilic polyethylene glycol block with single molecular weight, and contains acyl azide functional groups, and the yield is 80% -85%.
The compound shown in the formula C5 is characterized by nuclear magnetic hydrogen spectrum and mass spectrum, and the results are shown in figures 3 and 4.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by formula C5 prepared according to example 1 of the present invention.
FIG. 4 is a mass spectrum of a compound represented by formula C5 prepared according to example 1 of the present invention.
Referring to fig. 3 and 4, nuclear magnetic resonance hydrogen spectrum and mass spectrum test results show that the compound shown as the formula C5 containing acyl azide functional groups and having hydrophilic polyethylene glycol blocks with single molecular weight is prepared.
HydrophilicThe synthetic route of the molecule is shown below:
(2) From the slaveThe end starts to be connected with degradable construction element
The compound (1.0 g,1.32 mmol) represented by formula C5 was dissolved in 10mL of anhydrous toluene, azeotropically dehydrated, and then 10mL of anhydrous toluene was added. After reaction for 6h at 85℃under nitrogen, it was returned to room temperature. Another flask was taken, and after azeotropic dehydration of the compound of formula C6 (0.39 g,1.32 mmol) and 0.5% dibutyltin laurate (DBTL) catalyst with 10mL of toluene, 10mL of anhydrous Tetrahydrofuran (THF) was added for dissolution, and the mixture was transferred to a flask containing the compound of formula C5, and the reaction system was reacted at room temperature for 8 to 12 hours. After the reaction is finished, the solvent is removed by rotation, petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain a corresponding product, which is marked as a compound shown as a formula C7, and the yield is 80-85%.
The compound shown in the formula C7 after being connected with the degradable building element is characterized by nuclear magnetic hydrogen spectrum and mass spectrum, and the results are shown in figures 5 and 6.
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by formula C7 prepared according to example 1 of the present invention.
FIG. 6 is a mass spectrum of a compound represented by formula C7 prepared according to example 1 of the present invention.
Referring to fig. 5 and 6, characterization results of nuclear magnetic hydrogen spectrum and mass spectrum show that the compound shown as the formula C7 is prepared.
From the slaveThe synthetic route for the end-initiated access to the degradable building element is as follows:
according to the embodiment of the invention, the compound shown as the formula C5 and the compound shown as the formula C6 containing hydroxyl react, namely, the isocyanate generated after heating the acyl azide and the hydroxyl react efficiently.
(3) Containing trigger elementsAnd (3) accessing the terminal:
a compound (2.0 g,1.94 mmol) represented by the formula C7, a compound (1.75 g,1.94 mmol) represented by the formula C8 and 0.5% dibutyl tin laurate (DBTL) catalyst were azeotropically dehydrated with 10mL of toluene, then 10mL of anhydrous N-methylpyrrolidone (NMP) was added for dissolution, and the reaction system was reacted at 85℃for 5 to 10 hours under nitrogen atmosphere. After the reaction is finished, 50mL of ethyl acetate is added into the system, the system is washed twice by saturated saline water, anhydrous sodium sulfate is added for drying, the solvent is removed by reduced pressure rotary evaporation, and petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain a corresponding product, which is marked as a compound shown as a formula C9, and the yield is 55-75%. Wherein the compound shown as the formula C9 is a cationic lipid precursor.
A compound (2.0 g,1.09 mmol) represented by formula C9, a compound (0.3 g,1.31 mmol) represented by formula C10 and benzoin dimethyl ether (DMPA, 0.28g,1.09 mmol) as catalysts were added to a 25 mL-tube, 10mL of anhydrous Tetrahydrofuran (THF) was added as a solvent, and after deoxygenation, the mixture was irradiated with 365-UV-curing lamps at room temperature for 2-8 hours. Reaction junctionAfter the beams are bundled, the solvent is removed by rotation, and the petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain the corresponding product, which is marked as a compound shown as a formula C11, and the yield is 50-70%. Wherein the compound shown as the formula C10 is a material containing a cationic tertiary amine with a sulfhydryl end and an R group, and the compound shown as the formula C11 is an access triggering elementAnd a compound having a trityl end after the terminal is modified with an R group.
The compound (2.1 g,1.02 mmol) represented by formula C11 was added to a single-necked flask, and 80% acetic acid (20 mL) was added thereto as a solvent to react at room temperature for 6 hours. After the reaction is finished, the solvent is removed by rotation, and the product is washed by petroleum ether for 3 times, so that the corresponding product is obtained, wherein the compound shown in the formula C12 is obtained, the compound shown in the formula C12 is the cationic lipid shown in the formula (I), and the yield is 50-75%. Wherein the compound shown as C12 is an access trigger motifAnd a compound having a hydroxyl end after the terminal is modified with an R group.
The compound represented by formula C11 was characterized by mass spectrometry and the results are shown in fig. 7.
Referring to FIG. 7, the mass spectrum characterization result shows that the compound shown as the formula C11 is prepared.
The compound of formula C12 was characterized by mass spectrometry and the results are shown in fig. 8.
Referring to FIG. 8, the mass spectrum characterization result shows that the compound shown as the formula C12 is prepared.
Accessing a message containing a trigger elementThe synthetic route for preparing cationic lipids using cationic lipid precursors is shown below:
(4) Preparing a nanoparticle assembly by adopting a microfluidic technology:
the cationic lipid shown in formula C12, PEG-lipid and cholesterol ethanol solution and mRNA-citric acid buffer solution are filtered through a 0.22 mu m filter membrane, then sucked into a syringe and connected with a sample inlet pipe of a microfluidic device, the flow rates of the two sample pipes are set, the flow rate of the lipid ethanol solution is 2400 mu L-3000 mu L, and the flow rate of the mRNA-citric acid solution is 8000 mu L-9000 mu L. Dialysis and particle size testing were performed with an encapsulation efficiency of about 95%. The particle size was characterized by TEM, and the results are shown in fig. 9A to 9B.
Referring to fig. 9A and 9B, TEM characterization results show that nanoparticle assemblies with uniform particle sizes are assembled.
Example 2
The raw materials used in the examples are described below: benzoin dimethyl ether (DMPA), diphenyl azide phosphate (DPPA), triethylamine (TEA) and N-methylpyrrolidone (NMP) were purchased from Sigma-Aldrich company and used without further purification. Tetrahydrofuran was dried over sodium wire and refluxed, and distilled for use. Other reagents such as petroleum ether, ethyl acetate, dibutyl tin laurate, potassium carbonate, methanol and the like are all analytically pure and are purchased from national pharmaceutical chemicals company, inc., and are used without further purification. The ultrapure water used in the experiment was prepared by a Milli-QSP intelligent ultrapure water system, and the resistivity was 18.4MΩ & cm.
In the present embodiment, n is 3,a1, a is 1, R 1 is-H, R 2 Is->Or H, R 3 ～R 7 is-H->P1, b is 8, X is-H, R isN1 or N2, c is 1, d is 1, and e is 1.
(1) Accessing two degradable building element molecules
The compound of formula C7 (1.0 g,0.97 mmol), p-hydroxymethylbenzoic acid (0.148, 0.97 mmol) and 0.5% DBTL catalyst were dissolved in 10mL of anhydrous toluene and azeotropically dehydrated before adding 10mL of anhydrous toluene. Reacting for 8-12 h in nitrogen atmosphere at 85 ℃. After the reaction is finished, the solvent is removed by rotation, and the petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain the corresponding product, which is marked as C13 compound, and the yield is 75-85%.
The structure of the compound shown in formula C13 was characterized by mass spectrometry, and the results are shown in fig. 10. Referring to fig. 10, the mass spectrometry test results indicate that the compound represented by formula C13 was prepared.
The compound (2.00 g,1.84 mmol) represented by the formula C13 and triethylamine (0.86 g,1.84 mmol) were dissolved in 20mL THF, DPPA (0.506 g,1.84 mmol) was added dropwise at room temperature, the reaction was stirred at room temperature for 2 to 18 hours, after the completion of the reaction, the solvent was removed by rotation, and the corresponding product was isolated and purified by column chromatography using petroleum ether-ethyl acetate as a eluent, and was recorded as C14 compound in a yield of 80 to 85%.
A compound of formula C14 (1.5 g,1.35 mmol), a compound of formula C8 (1.21 g,1.35 mmol) and 0.5% DBTL catalyst were dissolved in 10mL of anhydrous toluene and azeotropically dehydrated, followed by the addition of 10mL of anhydrous toluene. Reacting for 8-12 h in nitrogen atmosphere at 85 ℃. After the reaction is finished, the solvent is removed by rotation, and the petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain the corresponding product, which is marked as C15 compound, and the yield is 65-85%. Wherein the compound shown as the formula C15 is a cationic lipid precursor.
The compound of formula C15 was characterized by mass spectrometry and the results are shown in figure 11. Referring to fig. 11, the mass spectrometry test results indicate that the compound represented by formula C15 was prepared.
The synthetic route is as follows:
(2) Introduction of a tertiary amine group R (R is N1):
a compound (2.0 g,1.01 mmol) represented by formula C15, a compound (0.28 g,1.21 mmol) represented by formula C10 and a DMPA (0.299 g,1.01 mmol) catalyst were added to a 25 mL-tube sealer, 10mL of anhydrous THF was added as a solvent, and after oxygen removal, the mixture was irradiated with 365 UV curing lamp at room temperature for 2 to 8 hours. After the reaction is finished, the solvent is removed by rotation, and the petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain the corresponding product, which is marked as C16 compound, and the yield is 50-70%.
Wherein the compound shown as the formula C10 is a material containing a cationic tertiary amine with a sulfhydryl end and an R group, and the compound shown as the formula C16 is an access triggering elementAnd the end of the compound adopts a compound with a trityl end after the R group is N1 modified with cations. It has two degradable motifs but contains only one R group.
The compound (2.23 g,1.01 mmol) represented by formula C16 was added to a single-necked flask, and 20mL of 80% acetic acid was added thereto as a solvent to react at room temperature for 6 hours. After the reaction, the solvent was removed by spin and the product was washed 3 times with petroleum ether to give the corresponding product, designated as C17 compound, in 50 to 75% yield. The compound shown as the formula C17 is the cationic lipid shown as the formula (I), and the yield is 50-75%. Wherein the compound shown as C17 is an access trigger motifAnd a compound having a hydroxyl end after the terminal is modified with an R group.
The compound of formula C17 was characterized by mass spectrometry and the results are shown in figure 12. Referring to fig. 12, the mass spectrometry test results indicate that the compound represented by formula C17 was prepared.
The synthetic route is as follows:
(3) Introduction of a tertiary amine group R (R is N2):
a compound (2.0 g,1.01 mmol) represented by formula C15, a compound (0.3 g,1.21 mmol) represented by formula C18 and a DMPA (0.259 g,1.01 mmol) catalyst were added to a 25mL tube sealer, 10mL of anhydrous THF was added as a solvent, and after oxygen removal, the mixture was irradiated with 365 UV curing lamps at room temperature for 2 to 8 hours. After the reaction is finished, the solvent is removed by rotation, and the petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain the corresponding product, which is marked as C19 compound, and the yield is 50-70%.
Wherein the compound shown as the formula C18 is a material containing a cationic tertiary amine with a sulfhydryl end and an R group, and the compound shown as the formula C19 is an access triggering elementAnd the end of the compound adopts a compound with a trityl end after the R group is N2 modified with positive ions. It has two degradable motifs but contains only one R group.
The compound (2.25 g,1.01 mmol) represented by formula C19 was added to a single-necked flask, and 20mL of 80% acetic acid was added thereto as a solvent to react at room temperature for 6 hours. After the reaction, the solvent was removed by spin and the product was washed 3 times with petroleum ether to give the corresponding product, designated as C20 compound, in 50% to 75% yield. Wherein the compound shown as C20 is an access trigger motifAnd a compound having a hydroxyl end after the terminal is modified with an R group.
The compound shown in formula C20 was characterized by mass spectrometry and the results are shown in fig. 13. Referring to fig. 13, mass spectrometry results indicate that the compound of formula C20 was prepared.
The synthetic route is as follows:
(4) Preparing a nanoparticle assembly by adopting a microfluidic technology:
the cationic lipid represented by formula C17 or the ethanol solution of PEG-lipid and cholesterol represented by formula C20 and the mRNA-citric acid buffer solution were filtered through a 0.22 μm filter membrane, and then sucked into a syringe and connected to a microfluidic device sample introduction tube, and the flow rates of the two sample tubes were set, and the flow rate of the lipid ethanol solution was set to 2400. Mu.L to 3000. Mu.L and the flow rate of the mRNA-citric acid solution was set to 8000. Mu.L to 9000. Mu.L. The encapsulation efficiency of the system using the compound of formula C17 and the compound of formula C20 as cationic lipids was about 95% and 97%, respectively.
Comparative example 1
The raw materials used are as follows: diphenyl azide phosphate (DPPA), triethylamine (TEA) and N-methylpyrrolidone (NMP) were purchased from Sigma-Aldrich company and used without further purification. Tetrahydrofuran was dried over sodium wire and refluxed, and distilled for use. Other reagents such as petroleum ether, ethyl acetate, dibutyl tin laurate, potassium carbonate, methanol and the like are all analytically pure and are purchased from national pharmaceutical chemicals company, inc., and are used without further purification. The ultrapure water used in the experiment was prepared by a Milli-QSP intelligent ultrapure water system, and the resistivity was 18.4MΩ & cm.
In the present embodiment, n is 1,a1, a is 1, R 5 ～R 7 is-H->P1, b is 8, and X is-H.
(1) Preparation of control molecules with pendant tertiary amine-free groups
A compound of formula C8 (1.21 g,1.35 mmol), a compound of formula C5 (1.02 g,1.35 mmol) and 0.5% DBTL catalyst were dissolved in 10mL of anhydrous toluene and azeotropically dehydrated, followed by the addition of 20mL of anhydrous toluene. Reacting for 8-12 h in nitrogen atmosphere at 85 ℃. After the reaction is finished, the solvent is removed by rotation, and the petroleum ether-ethyl acetate is used as eluent for column chromatography separation and purification to obtain the corresponding product, which is marked as C21 compound, and the yield is 74-86%.
The compound (1.7 g,1.04 mmol) represented by formula C21 was added to a single-necked flask, and 20mL of 80% acetic acid was added thereto as a solvent to react at room temperature for 6 hours. After the reaction, the solvent was removed by spin and the product was washed 3 times with petroleum ether to give the corresponding product, designated as C22 compound, in 55% to 75% yield. Wherein the compound shown as C22 is an access trigger motifA compound having a hydroxyl end which is terminal and free of modification by an R group.
The compound shown in formula C22 was characterized by mass spectrometry and the results are shown in fig. 14. Referring to fig. 14, the mass spectrometry test results indicate that the compound represented by formula C22 was prepared.
The synthetic route is as follows:
(2) Preparing a nanoparticle assembly by adopting a microfluidic technology:
the cationic lipid shown in formula C22, PEG-lipid and cholesterol ethanol solution and mRNA-citric acid buffer solution are filtered through a 0.22 mu m filter membrane, then sucked into a syringe and connected with a sample inlet pipe of a microfluidic device, the flow rates of the two sample pipes are set, the flow rate of the lipid ethanol solution is 2400 mu L-3000 mu L, and the flow rate of the mRNA-citric acid solution is 8000 mu L-9000 mu L. The encapsulation efficiency of the system using the compound of formula C22 instead of cationic lipid was 28% which is significantly lower than that of the tertiary amine containing system.
(3) Comparison of responsive results
Nanoparticle assemblies prepared using the compound shown in formula C12, the compound shown in formula C17, the compound shown in formula C20, and the compound shown in formula C22 as cationic lipids using microfluidics were diluted to 0.1g/L, respectively, and placed in a buffer solution having a pH of 5.0 containing 2mM hydrogen peroxide, a buffer solution having a pH of 7.4 containing 2mM hydrogen peroxide, a buffer solution having a pH of 5.0 not containing 2mM hydrogen peroxide, and a buffer solution having a pH of 7.4 not containing 2mM hydrogen peroxide, respectively. Dynamic light scattering is then used to monitor the change in scattered light intensity and thereby track the degradation process of the nanoparticle.
Fig. 15A to 15D are characterization results of the comparative test for responsiveness according to example 3 of the present invention.
Referring to fig. 15A-15D, dynamic light scattering demonstrates that acid and reactive oxygen species dual triggering provides the nanoparticle assembly with the greatest degradation rate. In the figure, the abscissa represents incubation time, and the ordinate represents normalized scattering intensity.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (10)
1. A cationic lipid, characterized in that the cationic lipid has a structure represented by formula (I) or formula (II):
wherein n is more than or equal to 1 and less than or equal to 10;
a hydrophilic polyethylene glycol block having a single molecular weight;
is a hydrophobic block, the end of the hydrophobic block is C 2 ～C 21 Saturated or unsaturated alkyl chains of (a);
R 1 ～R 7 are each independently selected from-H, halogen atom, -CH 3 、-OCH 3 、
R of at least one of the n repeating units 1 、R 2 、R 3 And R is 4 At least one of the choices
R has a tertiary amine group and is bonded to the propoxy group by a thioether bond to form a group
2. The cationic lipid according to claim 1, wherein,selected from any one of structures shown in formulas (A1) to (A5):
wherein a is more than or equal to 1 and less than or equal to 18;
R 8 and R is 9 Are independently selected from-H, -OCH 3 、C 1 ～C 20 Alkyl, C of (2) 3 ～C 20 Cycloalkyl, C 2 ～C 20 Alkylene or C of (2) 2 ～C 20 Is a propargyl group of (c).
3. The cation of claim 1A daughter lipid, characterized in that,selected from any one of structures shown in formulas (P1) to (P12):
wherein b is more than or equal to 5 and less than or equal to 45,5, m is more than or equal to 45;
R 10 、R 11 are each independently selected from-H, halogen atom, -CH 3 ；
X is selected from any one of the structures shown below:
wherein h is more than or equal to 1 and less than or equal to 18.
4. The cationic lipid according to claim 1, wherein R is selected from any one of structures represented by formulae (N1) to (N6):
wherein c is more than or equal to 0 and less than or equal to 14, d is more than or equal to 0 and less than or equal to 16,0, e is more than or equal to 7, and f is more than or equal to 0 and less than or equal to 17.
5. The process of claim 4, wherein c is 1 or 2, d is 1 or 2, and e is 1 or 2.
6. A method of preparing a cationic lipid according to any one of claims 1 to 5, comprising:
carrying out addition reaction on a cationic lipid precursor and a compound RH with a sulfhydryl terminal and a tertiary amine group to obtain the cationic lipid with a structure shown in a formula (I) or a formula (II);
the structure of the cationic lipid precursor is shown as a formula (I-1) or a formula (II-1):
wherein n is more than or equal to 1 and less than or equal to 10;
a hydrophilic polyethylene glycol block having a single molecular weight;
is composed of one or two C 2 ～C 21 A hydrophobic block of alkyl chains;
R 1 ’～R 7 ' are independently selected from the group consisting of-H, halogen atoms, -CH 3 、-OCH 3 Or (b)
R of at least one of the n repeating units 1 ’、R 2 ’、R 3 ' and R 4 At least one of the choices
R has a tertiary amine group and forms a sulfhydryl terminus with H.
7. The method of claim 6, wherein the conditions of the addition reaction include: under the action of a catalyst, carrying out the addition reaction under the irradiation of ultraviolet light and at the heating temperature of 25-45 ℃;
wherein the catalyst comprises benzoin dimethyl ether; the irradiation duration of the ultraviolet irradiation is 2-8 hours.
8. A nanoparticle assembly, comprising:
the cationic lipid according to any one of claims 1 to 5;
at least one nucleic acid species, associated with the cationic lipid, forms the nanoparticle assembly by self-assembly.
9. A method of preparing the nanoparticle assembly of claim 8, comprising:
and combining the cationic lipid, PEG-lipid, cholesterol and nucleic acid substances by utilizing a microfluidic or nano flash precipitation technology, and obtaining the nanoparticle assembly through self-assembly.
10. Use of a cationic lipid according to any one of claims 1 to 5 in the manufacture of a medicament for nucleic acid delivery.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311048251.2A CN117126187A (en) | 2023-08-18 | 2023-08-18 | Cationic lipid, preparation method thereof, nanoparticle assembly and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311048251.2A CN117126187A (en) | 2023-08-18 | 2023-08-18 | Cationic lipid, preparation method thereof, nanoparticle assembly and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117126187A true CN117126187A (en) | 2023-11-28 |
Family
ID=88857691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311048251.2A Pending CN117126187A (en) | 2023-08-18 | 2023-08-18 | Cationic lipid, preparation method thereof, nanoparticle assembly and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117126187A (en) |
-
2023
- 2023-08-18 CN CN202311048251.2A patent/CN117126187A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9320713B2 (en) | Nanoparticles and nanoparticle compositions | |
| CN103588940B (en) | Amphipathilic block polymer, polymer vesicle and its preparation method and application | |
| CN105056213B (en) | A kind of supermolecule nano ball of glucose responding and its preparation method and application | |
| CN107641181B (en) | Diblock copolymer with light and pH dual responsiveness and preparation method thereof | |
| CA3145913A1 (en) | Nanomaterials containing constrained lipids and uses thereof | |
| CN108752594B (en) | Amphipathic block polymer based on azo reductase response and preparation method and application thereof | |
| CN110746599B (en) | UV (ultraviolet) light-responsive hyperbranched poly-beta-amino ester with high-efficiency gene delivery capacity as well as preparation method and application thereof | |
| CN102145173A (en) | Human serum albumin complex hydrophobically modified pullulan nanoparticles and preparation method thereof | |
| CN113461952B (en) | Active oxygen response type self-degradation polymer and preparation method and application thereof | |
| CN105463024A (en) | Multifunctional supramolecular gene delivery system based on polyethyleneimine-cyclodextrin and preparation method thereof | |
| WO2017063131A1 (en) | Cation polymer capable of removing positive charges by means of oxidative response, preparation method, and application | |
| CN110732027A (en) | stimulation response targeting polysaccharide supramolecular diagnosis and treatment assembly and preparation method thereof | |
| CN117126187A (en) | Cationic lipid, preparation method thereof, nanoparticle assembly and application | |
| JP4847318B2 (en) | Charged initiator polymers and methods of use | |
| CN109970831B (en) | Gemcitabine prodrug compound, bionic nano-drug carrier and preparation method thereof | |
| CN116574070A (en) | Multi-tail type ionizable lipid, and preparation method and application thereof | |
| CN114106348B (en) | Phenylboronic acid modified intracellular degradable hyperbranched poly (beta-amino ester), preparation method and protein delivery application thereof | |
| CN107049950B (en) | Preparation method of cyclodextrin polymer drug-loaded vesicle | |
| CN110642968B (en) | Double-enzyme responsive dumbbell-shaped super-amphiphilic molecule and preparation method and application thereof | |
| CN113248556A (en) | Assembly of nucleic acid grafted azobenzene, preparation method and application | |
| US8901092B2 (en) | Functionalized polysaccharides for active agent delivery | |
| CN114874353B (en) | Non-viral gene drug carrier based on chitosan oligosaccharide and preparation method thereof | |
| WO2008117300A2 (en) | Improved process for the preparation of cadexomer iodine | |
| CN114672031B (en) | PH responsive polymer nano-drug and preparation method thereof | |
| CN112717140B (en) | Preparation and application of HP1 gamma-containing guanidinated polyaminoamine polymer gene vector compound |
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 |