CN115304756A - Five-membered lipid nanoparticle and preparation method and application thereof - Google Patents
Five-membered lipid nanoparticle and preparation method and application thereof Download PDFInfo
- Publication number
- CN115304756A CN115304756A CN202210114678.7A CN202210114678A CN115304756A CN 115304756 A CN115304756 A CN 115304756A CN 202210114678 A CN202210114678 A CN 202210114678A CN 115304756 A CN115304756 A CN 115304756A
- Authority
- CN
- China
- Prior art keywords
- lipid
- fnp
- pbae
- membered
- peg
- 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.)
- Granted
Links
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 73
- 150000002632 lipids Chemical class 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 28
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 28
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 28
- -1 cationic lipid Chemical class 0.000 claims abstract description 20
- 229930182558 Sterol Natural products 0.000 claims abstract description 15
- 150000003432 sterols Chemical class 0.000 claims abstract description 15
- 235000003702 sterols Nutrition 0.000 claims abstract description 15
- 150000003904 phospholipids Chemical class 0.000 claims abstract description 14
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 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 18
- 238000000034 method Methods 0.000 claims description 14
- 235000012000 cholesterol Nutrition 0.000 claims description 9
- 239000012071 phase Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 claims description 6
- 239000000178 monomer Substances 0.000 claims description 6
- 239000008346 aqueous phase Substances 0.000 claims description 4
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims description 3
- DSNRWDQKZIEDDB-SQYFZQSCSA-N 1,2-dioleoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-SQYFZQSCSA-N 0.000 claims description 3
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 claims description 3
- WALUVDCNGPQPOD-UHFFFAOYSA-M 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCC WALUVDCNGPQPOD-UHFFFAOYSA-M 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- FVXDQWZBHIXIEJ-LNDKUQBDSA-N 1,2-di-[(9Z,12Z)-octadecadienoyl]-sn-glycero-3-phosphocholine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC FVXDQWZBHIXIEJ-LNDKUQBDSA-N 0.000 claims description 2
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 claims description 2
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 claims description 2
- 244000000626 Daucus carota Species 0.000 claims description 2
- 235000002767 Daucus carota Nutrition 0.000 claims description 2
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 claims description 2
- 238000006845 Michael addition reaction Methods 0.000 claims description 2
- 150000003973 alkyl amines Chemical class 0.000 claims description 2
- 125000002947 alkylene group Chemical group 0.000 claims description 2
- 125000003118 aryl group Chemical group 0.000 claims description 2
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol 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)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 claims description 2
- 235000000431 campesterol Nutrition 0.000 claims description 2
- 125000002837 carbocyclic group Chemical group 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 125000005842 heteroatom Chemical group 0.000 claims description 2
- 125000000623 heterocyclic group Chemical group 0.000 claims description 2
- 239000012931 lyophilized formulation Substances 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 125000000467 secondary amino group Chemical class [H]N([*:1])[*:2] 0.000 claims description 2
- 239000007858 starting material Substances 0.000 claims description 2
- 150000003512 tertiary amines Chemical class 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- WTBFLCSPLLEDEM-JIDRGYQWSA-N 1,2-dioleoyl-sn-glycero-3-phospho-L-serine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC WTBFLCSPLLEDEM-JIDRGYQWSA-N 0.000 claims 1
- PZNPLUBHRSSFHT-RRHRGVEJSA-N 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[C@@H](COP([O-])(=O)OCC[N+](C)(C)C)COC(=O)CCCCCCCCCCCCCCC PZNPLUBHRSSFHT-RRHRGVEJSA-N 0.000 claims 1
- 101001105486 Homo sapiens Proteasome subunit alpha type-7 Proteins 0.000 claims 1
- 102100021201 Proteasome subunit alpha type-7 Human genes 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- UMGXUWVIJIQANV-UHFFFAOYSA-M didecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCC[N+](C)(C)CCCCCCCCCC UMGXUWVIJIQANV-UHFFFAOYSA-M 0.000 claims 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims 1
- OIWCYIUQAVBPGV-DAQGAKHBSA-N {1-O-hexadecanoyl-2-O-[(Z)-octadec-9-enoyl]-sn-glycero-3-phospho}serine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC OIWCYIUQAVBPGV-DAQGAKHBSA-N 0.000 claims 1
- 210000004072 lung Anatomy 0.000 abstract description 53
- 238000004108 freeze drying Methods 0.000 abstract description 27
- 230000008685 targeting Effects 0.000 abstract description 12
- 239000002994 raw material Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000012377 drug delivery Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 77
- 238000001890 transfection Methods 0.000 description 56
- 108020004999 messenger RNA Proteins 0.000 description 55
- 230000000694 effects Effects 0.000 description 34
- 239000000203 mixture Substances 0.000 description 27
- 238000001727 in vivo Methods 0.000 description 25
- 239000000463 material Substances 0.000 description 18
- 108060001084 Luciferase Proteins 0.000 description 17
- 239000005089 Luciferase Substances 0.000 description 17
- 108010031318 Vitronectin Proteins 0.000 description 17
- 102100035140 Vitronectin Human genes 0.000 description 17
- 229920001223 polyethylene glycol Polymers 0.000 description 17
- 238000009472 formulation Methods 0.000 description 16
- 239000002245 particle Substances 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 238000011156 evaluation Methods 0.000 description 14
- 229920000642 polymer Polymers 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 11
- 238000005538 encapsulation Methods 0.000 description 11
- 210000003712 lysosome Anatomy 0.000 description 11
- 230000001868 lysosomic effect Effects 0.000 description 11
- 210000000056 organ Anatomy 0.000 description 11
- 239000002202 Polyethylene glycol Substances 0.000 description 10
- 238000009826 distribution Methods 0.000 description 9
- 238000006116 polymerization reaction Methods 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000002296 dynamic light scattering Methods 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 102000005962 receptors Human genes 0.000 description 8
- 108020003175 receptors Proteins 0.000 description 8
- 238000011529 RT qPCR Methods 0.000 description 7
- 125000002091 cationic group Chemical group 0.000 description 7
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 7
- 210000003734 kidney Anatomy 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 210000003462 vein Anatomy 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 108010020147 Protein Corona Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000002889 endothelial cell Anatomy 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 108010054624 red fluorescent protein Proteins 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 206010018910 Haemolysis Diseases 0.000 description 4
- 229920001212 Poly(beta amino esters) Polymers 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000008588 hemolysis Effects 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 102000006495 integrins Human genes 0.000 description 4
- 108010044426 integrins Proteins 0.000 description 4
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000004700 cellular uptake Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000005556 structure-activity relationship Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- OOGKYHLYXDOJPR-JDVCJPALSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCC\C=C/CCCCCCCC OOGKYHLYXDOJPR-JDVCJPALSA-M 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102000013918 Apolipoproteins E Human genes 0.000 description 2
- 108010025628 Apolipoproteins E Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CXRFDZFCGOPDTD-UHFFFAOYSA-M Cetrimide Chemical compound [Br-].CCCCCCCCCCCCCC[N+](C)(C)C CXRFDZFCGOPDTD-UHFFFAOYSA-M 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 2
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 102000000853 LDL receptors Human genes 0.000 description 2
- 108010001831 LDL receptors Proteins 0.000 description 2
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 230000010799 Receptor Interactions Effects 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 210000000709 aorta Anatomy 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 108010007093 dispase Proteins 0.000 description 2
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 229960004956 glycerylphosphorylcholine Drugs 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 230000003908 liver function Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000002132 lysosomal effect Effects 0.000 description 2
- 108700021021 mRNA Vaccine Proteins 0.000 description 2
- 229940126582 mRNA vaccine Drugs 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 2
- 230000002685 pulmonary effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- JFBCSFJKETUREV-UHFFFAOYSA-N 1,2 ditetradecanoylglycerol Chemical compound CCCCCCCCCCCCCC(=O)OCC(CO)OC(=O)CCCCCCCCCCCCC JFBCSFJKETUREV-UHFFFAOYSA-N 0.000 description 1
- MWRBNPKJOOWZPW-NYVOMTAGSA-N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-NYVOMTAGSA-N 0.000 description 1
- WTJKGGKOPKCXLL-VYOBOKEXSA-N 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC WTJKGGKOPKCXLL-VYOBOKEXSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- OPNYPDPAVTYAEF-JDVCJPALSA-M 2,3-bis[(Z)-octadec-9-enoxy]propyl-(3-hydroxypropyl)-dimethylazanium bromide Chemical compound [Br-].C[N+](CC(COCCCCCCCC\C=C/CCCCCCCC)OCCCCCCCC\C=C/CCCCCCCC)(CCCO)C OPNYPDPAVTYAEF-JDVCJPALSA-M 0.000 description 1
- JQDSSKQQSCMUJR-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;bromide Chemical compound [Br-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC JQDSSKQQSCMUJR-LQDDAWAPSA-M 0.000 description 1
- CXLFKOXWMITIOY-UHFFFAOYSA-M 2,3-dihexadecoxypropyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCCCC CXLFKOXWMITIOY-UHFFFAOYSA-M 0.000 description 1
- NUHKVXHHVAYULW-UHFFFAOYSA-M 2,3-dioctadecoxypropyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCCCCCC NUHKVXHHVAYULW-UHFFFAOYSA-M 0.000 description 1
- UBOKASXZHPZFRZ-UHFFFAOYSA-N 2-phenyl-1h-indole-4,6-diamine Chemical compound N1C2=CC(N)=CC(N)=C2C=C1C1=CC=CC=C1 UBOKASXZHPZFRZ-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 239000012114 Alexa Fluor 647 Substances 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 108050007222 Coronin Proteins 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- XULFJDKZVHTRLG-JDVCJPALSA-N DOSPA trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCNC(=O)C(CCCNCCCN)NCCCN)OCCCCCCCC\C=C/CCCCCCCC XULFJDKZVHTRLG-JDVCJPALSA-N 0.000 description 1
- GZDFHIJNHHMENY-UHFFFAOYSA-N Dimethyl dicarbonate Chemical compound COC(=O)OC(=O)OC GZDFHIJNHHMENY-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- 102100022337 Integrin alpha-V Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 1
- 208000035977 Rare disease Diseases 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 235000011449 Rosa Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 108010048673 Vitronectin Receptors Proteins 0.000 description 1
- 241001441550 Zeiformes Species 0.000 description 1
- NJFCSWSRXWCWHV-USYZEHPZSA-N [(2R)-2,3-bis(octadec-1-enoxy)propyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCC=COC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC=CCCCCCCCCCCCCCCCC NJFCSWSRXWCWHV-USYZEHPZSA-N 0.000 description 1
- HIHOWBSBBDRPDW-PTHRTHQKSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] n-[2-(dimethylamino)ethyl]carbamate Chemical compound C1C=C2C[C@@H](OC(=O)NCCN(C)C)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 HIHOWBSBBDRPDW-PTHRTHQKSA-N 0.000 description 1
- DFKXLISURAZMNN-JDVCJPALSA-M [Br-].C[N+](CC(COCCCCCCCC\C=C/CCCCCCCC)OCCCCCCCC\C=C/CCCCCCCC)(CCCCCO)C Chemical compound [Br-].C[N+](CC(COCCCCCCCC\C=C/CCCCCCCC)OCCCCCCCC\C=C/CCCCCCCC)(CCCCCO)C DFKXLISURAZMNN-JDVCJPALSA-M 0.000 description 1
- TUOOCVNZWZRZJP-JDVCJPALSA-M [Br-].C[N+](CC(COCCCCCCCC\C=C/CCCCCCCC)OCCCCCCCC\C=C/CCCCCCCC)(CCCCO)C Chemical compound [Br-].C[N+](CC(COCCCCCCCC\C=C/CCCCCCCC)OCCCCCCCC\C=C/CCCCCCCC)(CCCCO)C TUOOCVNZWZRZJP-JDVCJPALSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007259 addition reaction Methods 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 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
- 210000001367 artery Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 150000001783 ceramides Chemical class 0.000 description 1
- 238000012668 chain scission Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 102000018123 coronin Human genes 0.000 description 1
- KRQUGYHEWIVMJV-UHFFFAOYSA-N coronin Natural products CCCCCCCCCCCCC=C/CCC1OC1CCC2OC2CCCCCCCCCCC3=CC(C)OC3=O KRQUGYHEWIVMJV-UHFFFAOYSA-N 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 150000001982 diacylglycerols Chemical class 0.000 description 1
- 125000005265 dialkylamine group Chemical class 0.000 description 1
- 150000001985 dialkylglycerols Chemical class 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- PNZDZRMOBIIQTC-UHFFFAOYSA-N ethanamine;hydron;bromide Chemical compound Br.CCN PNZDZRMOBIIQTC-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- UHUSDOQQWJGJQS-UHFFFAOYSA-N glycerol 1,2-dioctadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(CO)OC(=O)CCCCCCCCCCCCCCCCC UHUSDOQQWJGJQS-UHFFFAOYSA-N 0.000 description 1
- BEBCJVAWIBVWNZ-UHFFFAOYSA-N glycinamide Chemical compound NCC(N)=O BEBCJVAWIBVWNZ-UHFFFAOYSA-N 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000012750 in vivo screening Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- HSMPDPBYAYSOBC-UHFFFAOYSA-N khellin Chemical compound O1C(C)=CC(=O)C2=C1C(OC)=C1OC=CC1=C2OC HSMPDPBYAYSOBC-UHFFFAOYSA-N 0.000 description 1
- 210000005246 left atrium Anatomy 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000004924 lung microvascular endothelial cell Anatomy 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- VYNDHICBIRRPFP-UHFFFAOYSA-N pacific blue Chemical compound FC1=C(O)C(F)=C2OC(=O)C(C(=O)O)=CC2=C1 VYNDHICBIRRPFP-UHFFFAOYSA-N 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000008103 phosphatidic acids Chemical class 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 230000004088 pulmonary circulation Effects 0.000 description 1
- 210000003492 pulmonary vein Anatomy 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 210000005092 tracheal tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
- C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
- C08G63/6854—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen derived from polycarboxylic acids and polyhydroxy compounds
- C08G63/6858—Polycarboxylic acids and polyhydroxy compounds in which at least one of the two components contains aliphatic unsaturation
-
- 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
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/91—Polymers modified by chemical after-treatment
- C08G63/914—Polymers modified by chemical after-treatment derived from polycarboxylic acids and polyhydroxy compounds
- C08G63/918—Polycarboxylic acids and polyhydroxy compounds in which at least one of the two components contains aliphatic unsaturation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Nanotechnology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a five-membered lipid nanoparticle and a preparation method and application thereof. The raw materials of the pentabasic lipid nanoparticle comprise cationic lipid, poly (beta-amino ester), sterol, phospholipid, PEG lipid and nucleic acid molecules. The pentabasic lipid nanoparticle has high stability, can be stored for a long time at 4 ℃ after freeze drying, and can be used as a nucleic acid drug delivery system (such as lung targeting and the like); has good tolerance and has a suitable surface zeta potential of approximately +5 mV.
Description
Technical Field
The invention relates to the technical field of chemical pharmacy and biology, relates to a nucleic acid delivery system, and particularly relates to a preparation method of a high-stability lung-targeted polymer-lipid nanoparticle and application of the high-stability lung-targeted polymer-lipid nanoparticle in nucleic acid delivery.
Background
The outbreak of new coronary pneumonia allows people to be aware of the severity of pulmonary infectious diseases. Besides infectious diseases, other related diseases of the lung, such as lung cancer and hereditary rare diseases, also lack effective treatment means. During new coronary pneumonia, a novel mRNA vaccine encapsulated in Lipid Nanoparticles (LNPs) showed up to 95% protective efficacy, the first mRNA vaccine to obtain FDA emergency use approval. In addition, lipid Nanoparticle (LNP) -based mRNA can radically cure genetic diseases by encoding a gene editing tool, and can also achieve immunotherapy of tumors by encoding tumor antigens, immune checkpoint inhibitors, bispecific antibodies, and the like. Therefore, the application of mRNA-LNP formulations for the treatment of lung-related diseases is undoubtedly of great potential and significance.
At present, a few studies propose mRNA nucleic acid lung targeting strategies, for example, SORT proposes that positively charged nanoparticles can realize lung delivery. However, the current use of LNP still faces a number of problems, where storage stability is a major drawback limiting its widespread clinical use and LNP formulations with low stability present a significant economic barrier to storage and transport. LNP instability is therefore a problem that is urgently sought to be solved. The main factors currently affecting the instability of mRNA-LNPs are the instability of mRNA and the instability of LNP:
1) Chemical components in mRNA are susceptible to oxidation and hydrolysis in the presence of water. For example, 2'OH groups on phosphate linkages in mRNA attack the p-o5' ester linkages, resulting in mRNA chain scission. This process requires water and can be catalyzed by either acids or bases. 2) Due to degradation of the chemical material, the force of the mRNA with the chemical lipid weakens and the mRNA leaks. Leaked naked mRNA is difficult to absorb by cells and can be rapidly degraded; 3) Due to insufficient repulsion between nanoparticles in water, lipid nanoparticles have a tendency to aggregate, fuse and leak.
Disclosure of Invention
In order to function effectively in vivo, lipid nanoparticle-mRNA needs to overcome a variety of extracellular and intracellular barriers. First, mRNA needs to be successfully encapsulated by lipid nanoparticles. Second, the lipid nanoparticles need to reach the target tissue and then be internalized by the target cell. Most importantly, the mRNA must escape the endosome and be translated into a functional protein. In addition, the preparation can be stored for a long period of time at 4 ℃ in clinical use, so as to reduce the economic burden during storage in transportation.
In general, the physicochemical properties of the nanoparticles are the basis for subsequent evaluation of effectiveness. For example, higher encapsulation efficiency of nucleic acids is a key factor in determining efficient transfection and appropriate particle size; zeta potential also affects its toxicity and its ability to disperse in vivo to some extent. The higher the Zeta potential, the greater the hemolytic capacity of erythrocytes under physiological conditions and the greater the toxicity. While PDI reflects the uniformity and completeness of the prescription to some extent. The proper particle size and potential of the nanoparticles can reduce the probability of embolism in vivo, promote the penetration of deep tissues in vivo and improve the uptake and transfection efficiency of cells.
The inventors have innovatively found that the addition of PBAE and cationic lipids can increase the stability of FNP.
For example, the present invention employs the classical cationic lipid DOTAP, which has a strong positive charge to prevent intermolecular aggregation; secondly, an auxiliary polymer poly beta-amino ester (PBAE) is introduced into the traditional four-component cation LNP formula for the first time to form a novel five-membered nanoparticle FNP. PBAE is used as a protonized cationic material containing multiple diazo, thereby further enhancing the encapsulation of mRNA, preventing mRNA leakage, reducing adverse effects caused by crystallization and vacuum dehydration in the freeze-drying process and further ensuring the stability of the nanoparticles. The addition of the two components ensures that the nanoparticles have high stability and can well endure the freeze-drying process, and further, the influence of moisture on the hydrolysis of mRNA and lipid materials can be removed through freeze-drying.
The invention constructs a novel PBAE polymer library with different side chain lengths and different polymerization degrees, and further discusses the structure-activity relationship between the PBAE polymer library in vivo and in vitro. The closer the carbon chain length of the PBAE is to C16, the higher the delivery efficiency is; the higher the degree of polymerization of the PBAE, the higher the delivery efficiency. The specific and efficient lung targeting PBAE material is screened out by FNP of the novel auxiliary polymer based on the components in vivo, and the stabilization time at 4 ℃ after freeze drying exceeds 3 months.
In addition, the invention researches the protein crown composition of FNP by mass spectrometry and further researches the lung targeting mechanism of the FNP. It was found that vitronectin is highly enriched in FNP. The primary microvascular endothelial cells of mouse lung are firstly adopted to verify that the vitronectin passes through the lung endothelial cell integrin receptor alpha v β 3 Binding, mediates lung-specific targeting of FNP.
The invention mainly solves the technical problems through the following technical scheme.
The invention aims to provide a lung targeting nucleic acid delivery system which can effectively encapsulate nucleic acid, can be efficiently absorbed by cells and quickly escape out of lysosomes, and finally realizes efficient expression of target genes in vivo, and meanwhile, the system has high stability and can be stored for a long time at 4 ℃ after being frozen and dried.
In order to achieve the purposes of effectively encapsulating nucleic acid and improving cellular uptake and lysosome escape efficiency, the invention discloses the following technical scheme: a polymer-lipid nanoparticle is prepared by adding an auxiliary polymer PBAE and a cationic lipid into a lipid nanoparticle, wherein the nanoparticle is composed of the polymer PBAE, the cationic lipid, sterol, phospholipid, PEG lipid and one or more nucleic acid molecules, and the nucleic acid molecules are wrapped in the lipid and the polymer to form a layer-by-layer wrapped structure.
The invention introduces an auxiliary polymer poly beta-amino ester (PBAE) into a traditional four-component cation LNP formula for the first time to form a novel five-membered nanoparticle FNP. PBAE further enhances the encapsulation of mRNA as a protonatable polydiazon-containing cationic material.
The present invention provides a polymerized (beta-amino ester) (PBAE) comprising formula (I);
wherein:
R 1 independently a fatty chain with or without hydroxyl groups, R 2 Independently is an end group monomer comprising a primary, secondary or tertiary amine, R comprises a linear or branched C 1 -C 50 An alkylene chain which may further comprise one or more heteroatoms or one or more carbocyclic, heterocyclic or aromatic groups;
m is 1-15, n is 3-45, x is 1-13.
In the present invention, R is 1 Preferably independently is
The R is 2 Preferably independently is
Said R is preferably independently
The preparation of the PBAE may be conventional in the art, e.g. the preparation comprises: r is to be 1 、R 2 R and alkylamine are mixed together to perform the Michael addition reaction.
The invention also provides a pentameric lipid nanoparticle whose raw materials comprise a cationic lipid, a polymeric (beta-aminoester) as described above, a sterol, a phospholipid and a PEG lipid.
When the pentameric lipid nanoparticle is used as a nucleic acid delivery system, it may further comprise a nucleic acid molecule.
In the present invention, the molar ratio of the cationic lipid to the PBAE is preferably (2.5 to 40): 1.
In the present invention, the relative amount of the mass of the cationic lipid and the PBAE to the nucleic acid molecule is preferably (10 to 80): 1.
In the present invention, the molar ratio of the phospholipid to the sterol is preferably (0.2 to 1): 1.
in the present invention, the molar ratio of the phospholipid to the PEG lipid is preferably (5 to 20): 1.
In a preferred embodiment of the invention, the ratio of n to m is 7:3; namely: the proportion of fatty chains is 30%.
In the present invention, x is preferably 3 to 11.
In the present invention, the sterol is selected from one or more of cholesterol, campesterol, algal sterol, and carrot sterol; cholesterol is preferred.
In the context of the present invention, cationic lipids refer to permanently positively charged lipids, which may be selected from the list comprising: trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), dimethyl-2,3-dioleyloxypropyl-2- (2-spermimido) ethylammonium bromide (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORI), dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide (DORIE-HP), dimethyl-4-hydroxybutyl-2,3-dioleyloxypropylammonium bromide (DORIE-HB), dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide (DORIE-HPc), dimethyl-2-hydroxyethyl-2,3-dihexadecyloxypropylammonium bromide (DPRIE), dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide (DSRIE), dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide (DMRIE), N- (2-sperminoyl) -N', n ' -dioctadecyl glycinamide (DOGS), 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), 3 β - [ N- (N ', N ' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol).
The term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group, suitable phospholipids may be selected from the list comprising: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18, 1,2-docosahexanoyl-sn-glycerol-3-phosphocholine, 1,2-diphytanoyl-sn-glycerol-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinonoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-docosahexanoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPG) sodium salt (DOPG), sphingomyelin, and mixtures thereof.
The term "PEG lipid" or alternatively "pegylated lipid" refers to any suitable lipid modified with PEG (polyethylene glycol) groups. May be selected from the list comprising: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. More specific examples of such PEG lipids include C14-PEG2000 (1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG 2000)) and C18-PEG5000 (1,2-distearoyl-rac-glycerol, methoxypolyethylene glycol-5000 (DSG-PEG 5000)).
In a specific embodiment, the PEG lipid is methoxy polyethylene glycol-2000 (DMG-PEG 2000).
In the present invention, the nucleic acid molecule may be RNA and/or DNA; such as mRNA, pDNA.
In a preferred embodiment of the present invention, the method for preparing the five-membered lipid nanoparticle comprises:
dissolving a cationic lipid, a polymeric (. Beta. -aminoester) as described above, a sterol, a phospholipid, and a PEG lipid into an ethanol phase, and dissolving a nucleic acid molecule into an aqueous phase;
mixing the two phases to obtain the five-membered lipid nanoparticles;
the two phases are preferably mixed in a volume ratio of ethanol to water 1:1.
In a specific embodiment of the invention, the raw materials of the five-membered lipid nanoparticle comprise DOTAP, PBAE, cholesterol, DOPE and PEG lipid, and the molar ratio is (DOTAP + PBAE) DOPE: cholesterol: DMG-PEG = 50.
The invention also provides a pharmaceutical composition or vaccine comprising the five-membered lipid nanoparticle as described above and a pharmaceutically acceptable carrier.
The invention also provides the application of the polymerized beta-amino ester in the preparation of the five-membered lipid nanoparticle.
The present invention also provides a lyophilized formulation comprising the five-membered lipid nanoparticle as described above.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the PBAE prepared by the method has good biocompatibility. The five-membered lipid nanoparticles prepared by the method can realize high-efficiency and high-specificity in-vivo transfection, have high stability, can be stored for a long time at 4 ℃ after freeze drying, and can be used as a nucleic acid drug delivery system (such as lung targeting and the like); has good tolerance and has a suitable surface zeta potential of approximately +5 mV.
Drawings
FIGS. 1A-1C show PBAE structures, synthetic methods, and partially representative hydrogen spectra.
FIG. 2 shows PBAE delivery EGFP-mRNA transfection efficiency screening in 293T cells.
FIG. 3 is an evaluation of cell viability after incubation of FNP containing luciferase mRNA with 293T cells.
FIG. 4 shows the results of screening for FNP transfected luciferase mRNA in cells.
Figure 5 is an in vivo result of FNP delivering different doses of luciferase mRNA, the expression efficiency appeared dose-dependent (0.15-0.45 mg/kg data mean ± s.e.m. n = 3).
FIG. 6 is FNP in vivo transfection screen. Luciferase mRNA was dosed (0.3 mg/kg).
FIG. 7 is FNP delivering high doses of mRNA (0.6 mg/kg) well tolerated in vivo; the renal function (a, BUN, b, CREA) and liver function (c, AST, d, ALT) measurements all showed no apparent toxicity.
FIG. 8 shows the results of H & E staining of each major organ. FNP delivers high doses of mRNA (0.6 mg/kg) well tolerated in vivo; scale bar =50 μm.
FIG. 9 is a cryo-electron micrograph of FNP.
FIG. 10 shows the mRNA Encapsulation Efficiency (EE) of FNP.
FIG. 11 shows the particle size, PDI and potential of FNP.
FIG. 12 shows the evaluation of FNP for in vivo hemolysis under conditions of pH5.0 (simulated lysosome acidic environment) and pH7.4 (simulated plasma physiological environment).
Figure 13 is cellular uptake and lysosomal escape; 4,6-diamino 2-phenylindole (DAPI) stains the nucleus (blue) and LysoTracker stains the lysosome (green).
FIG. 14 shows the uptake of FNP into 293T cells.
Figure 15 is the efficiency of lysosomal escape of FNP in 293T cells.
FIG. 16 is a comparison of the effect of transfection of cells with conventional LNPs and after introduction of positive and negative charges and before and after lyophilization with PBAE alone.
FIG. 17 is a comparison of particle size, PDI and potential after conventional LNP and introduction of positive and negative charges and before and after lyophilization of PBAE alone formulations.
FIG. 18 shows that FNP after lyophilization (left panel) can maintain stable cell transfection effect for a long time at 4 ℃ while FNP without lyophilization decreases rapidly (right panel).
FIG. 19 shows the particle size, PDI and potential of freeze-dried FNP that remains stable at 4 ℃ for a long period of time.
FIG. 20 shows FNP induced specific tdTom fluorescent expression in the lungs of Ai9 mice.
FIG. 21 is a fluorescent microscope image showing lung tdTomato positive cells after FNP administration of cre mRNA; scale bar: 50 μm.
Fig. 22 is a graph quantifying the percentage of tdTom + cells in a particular cell type in the lung using flow cytometry (FACS).
FIG. 23 is an organ distribution of FNP; a) Organ distribution image of FNP harboring Cy5-mRNA, b) checking the mRNA distribution of each organ by qPCR.
FIG. 24 is vitronectin-rich protein corona mediated alpha v β 3 Receptor-associated lung-specific expression; a) Proteomic analysis of coronin isolated from 11C18E1 (n =23.64fnp, control, dlin-MC3 LNP) (n = 4/group), b) FNP loaded with EGFP-mRNA incubated with different amounts of vitronectin vs v β 3 Receptor-positive U87 cell lines and mouse primary pulmonary microvascular endothelial cells and alpha v β 3 Transfection of receptor-negative HepG2 cell lines at 50 μm scale, c) quantitative analysis of the proportion of eGFP-positive cells in the panels b.
FIG. 25 is a selection of PBAE delivery pDNA in 293T cell transfection efficiency.
FIG. 26 is a graph of FNP effectively encapsulating pDNA; the particle size, PDI and Zeta potential (a) were measured using Dynamic Light Scattering (DLS) and agarose gel electrophoresis results indicated that FNP was able to effectively encapsulate pDNA (b).
FIG. 27 shows that FNP (11C18E1 n = 23.64) can efficiently deliver pDNA (0.3 mg/kg) to the mouse lungs.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.
The present invention studies the transfection effects of cells before and after lyophilization of formulations with different surface potentials. Firstly, after the traditional formulas of C12-200 and Dlin-MC3 are lyophilized and redissolved, the transfection effect is obviously reduced, which indicates that the traditional liver targeting nanoparticles are difficult to achieve; tolerating the process of lyophilization. According to our previous hypothesis, the cationic lipid is added to the nanoparticles to make the surfaces of the nanoparticles positively charged so as to increase the repulsion among the particles and reduce the agglomeration of the particles in the freeze-drying process so as to keep the particles stable, and then 50% of DOTAP is added to the formulas of C12-200 and Dlin-MC3 so as to make the nanoparticles with the positively charged surfaces, and the results show that the two formulas can maintain the transfection effect basically unchanged after freeze-drying. The results achieved further showed that the incorporation was 50%: after the two types of nanoparticles with negative surfaces of PA are freeze-dried, the transfection effect is obviously reduced.
In addition, the present invention designed a series of PBAE materials with different hydrophobic chain lengths, and based on the results of previous screens, selected one of the groups (11C16E2 n = 24.11) with the best in vitro and in vivo efficacy, investigated whether individual PBAE prescriptions could maintain transfection efficacy after lyophilization by facilitating encapsulation of nucleic acids. The results show that PBAE can effectively increase the stability of nanoparticles.
Combining the results of the above two aspects, it is predicted that the formulation stability can be effectively increased by adding polymer PBAE and cationic lipid to LNP and that the transfection effect can be maintained for a long time after lyophilization. The present invention further carried out stability studies on five FNP formulations which showed the best in vivo effect, and measured the cell transfection effect at different time points after lyophilization, and the results showed that the former four groups of FNPs all showed better stability, while the fifth group, although having the highest surface potential, showed less stability as the ability to transfect cells decreased with time, which may be related to the fact that the length of the hydrophobic chain of PBAE was shorter than that of the remaining four groups, which also suggests that not the higher the surface potential, the better the stability of nanoparticles, and PBAE plays an extremely important role in doing so.
Example 1
The invention designs and synthesizes a series of PBAE materials, the skeleton structure of which is 11, and the invention combines the following figures 1A-1C: the first 1 refers to the structure of back bone (B), i.e., B1; the second 1 refers to the structure of side chain (S), i.e., S1). To further investigate the effect of the end-capping groups of various PBAEs on their efficiencies, we selected two groups of end-capping groups, i.e., E1 and E2. Based on the assumption that alkyl side chains of PBAE can promote mRNA encapsulation and lysosome escape, we designed different lengths of carbon chain alkyl side chains, 8, 10, 12, 14, 16 and 18 respectively, and the addition of hydrophobic chains of different lengths helps PBAE to encapsulate nucleic acids into a more stable structure, so that it can withstand the process of freeze-drying. Since it has been reported that the degree of polymerization of PBAE has a great influence on the transfection efficiency, 3 main types of polymerization degrees (hereinafter, n; hereinafter, unless otherwise specified, 30% of aliphatic chain) were set as one of the variables, and the polymerization degrees were 4 to 7, 9 to 12, and 17 to 26, respectively. A series of PBAEs were synthesized by the Michael (Michael) addition reaction as shown in fig. 1A to 1C: the bisacrylate backbone (B) monomer was mixed with the total side chain (S) monomers in a molar ratio of 1.1 (30% of the total side chain monomers were alkyl chain monomers) with B in excess to ensure that the BS based polymer was bisacrylate terminated. Stirring at 90 ℃ for different times to give PBAE of different molecular weights. The resulting polymer base was dissolved in tetrahydrofuran or Dimethylsulfoxide (DMSO), and then 10-fold equivalent of the terminal group (E) molecule was added and stirred at room temperature for 2 hours. The resulting polymer was then precipitated from the solution by addition of diethyl ether, washed twice with diethyl ether and dried under vacuum. The resulting polymer was dissolved in CDCl3 or d6-DMSO and its molecular weight was measured by 1H-NMR (Bruker 400 MHz).
EXAMPLE 2 transfection Effect of FNP on 293T cells prepared with different PBAE materials at different prescription ratios
1. Preparation of FNP nanoparticles coated with EGFP mRNA
FNP was prepared, comprising PBAE, DOTAP, chol, DOPE, DMG-PEG and nucleic acid molecules, with an average diameter of 60-160nm and a surface potential of 3-20 mV.
The preparation method comprises the following steps: dissolving DOTAP, PBAE, DOPE, chol and absolute ethyl alcohol into an ethanol phase according to a specific molar ratio, wherein the molar ratio of (DOTAP + PBAE)/DOPE/Chol/DMG-PEG is fixed to be 50/10/38.5/1.5. The molar ratio of DOTAP to PBAE varied from 2.5/1 to 40/1 depending on the type of PBAE. The aqueous phase was 100mM citrate buffer containing nucleic acids (PH = 3). The mass ratio of DOTAP + PBAE to mRNA was 20. The two phases are mixed rapidly with a volume ratio of ethanol to water of 1/1. Expression levels were observed under a fluorescent microscope after 24 hours.
2. MTT method for researching influence of FNP on 293T cell in vitro survival rate
To evaluate PBAE transfection efficiency in vitro, the present invention selects 293T cells as a model. PBAE is taken as a protonatable cationic material, and the optimal proportion of PBAE is different from that of DOTAP due to different structures. Therefore, to determine the optimal ratios, EGFP-mRNA was coated in FNP at different DOTAP-PBAE ratios (DOTAP: PBAE =2.5, 5:1, 10 1) and transfected into 293T cells in complete medium. Thereafter, the most appropriate DOTAP/PBAE ratio for each PBAE was determined and will be continued for subsequent in vivo and in vitro experiments (fig. 2).
For in vitro cell transfection, fresh FNP was directly diluted in DMEM complete medium; for in vivo delivery, prepared FNP was dialyzed against 3500MWCO dialysis membrane in PBS solution at 4 ℃ for 2h.
The influence of FNP on the in vitro survival rate of 293T cells is researched by an MTT method, after FNP nanoparticles are added for transfection for 24 hours (the relative dosage of the FNP nanoparticles and the cells is 150ng mRNA/hole; and the inoculation density of the cells in a 96-hole plate is 10) 4 one/mL), MTT (5 mg/mL in PBS,20ul, beyotime) was added and the cells were incubated at 37 ℃ for 4h. At the end of the incubation period, the cell culture medium was removed, 150. Mu.l DMSO (solarbio) was added and the absorbance was measured at 490nm using a microplate reader (SpectraMax i3, MD). Cell viability (%) was calculated using treated cells compared to untreated control cells.
As shown in fig. 3. Most of these PBAEs were not significantly cytotoxic. 77.77% of PBAE cell activity was greater than 80%.
Example 3 evaluation of dose dependence of transfection Effect of FNP formulations of several different PBAE materials transfection of luciferase mRNA in mice
To further explore the structure-activity relationship of PBAE, the present invention transfected 293T cells with luciferase (Fluc) mRNA and quantified luciferase activity, as shown in FIG. 4, the transfection trends of EGFP-mRNA and Fluc-mRNA were essentially the same. According to the analysis of the results, the blocking of E1 has better transfection effect compared with the blocking of E2. The higher the polymerization degree, the better the transfection effect, and the closer the carbon chain length is to 16, the better the transfection effect.
According to the mRNA transfection results of cells, the first three materials 11c14E1 n =17.41,11c14E2 n =17.41 and 11c18E1 n =23.64 with the best transfection effect are selected, and whether dose dependence exists in vivo in FNP consisting of PBAE is researched (like example 1, the first 1 refers to the structure of back bone (B), namely B1, the second 1 refers to the structure of side chain (S), namely S1, and n refers to the polymerization degree of PBAE calculated by a hydrogen spectrum, and C14 or C18 represents the length of a carbon chain alkyl side chain).
The nanoparticle preparation method was the same as the above example, and after the nanoparticle preparation was completed, the prepared FNP was dialyzed in PBS solution against 3500mwco dialysis membrane at 4 ℃ for 2 hours. Then the luciferase is injected into a mouse body through tail vein, 30mg/mL of luciferin potassium salt is injected into the abdominal cavity according to the dose of 150mg/kg after six hours of administration, and the luciferase expression condition is detected about ten minutes after the injection.
As shown in FIG. 5, all three materials had lung specificity and dose sensitivity in the range of 0.15mg/kg to 0.45mg/kg Fluc mRNA. Subsequent evaluation of all PBAE in vivo will be performed at a dose of 0.3 mg/kg.
PBAE with luciferase activity (RLU/Well) greater than 100,000 and various degrees of aggregation 11C10E1 were selected for in vivo screening as shown in a in FIG. 6. As can be seen from b in 6, the 4 most efficient PBAE bioluminescent signals (11C16E2 n =24.11,11c18e1 n =23.64,11c16e1 n =11.85,11c14e2 n =17.41) are all mainly distributed in the lungs, with the best materialThe highest fluorescence efficiency of 1116E2 n =24.11 can reach 10 8 . In addition, the in vivo structure-activity relationship of PBAE is essentially identical to that in vitro. From the viewpoint of the degree of polymerization, 11C10E1 n =5.13,11C10E1 n =9.66, and 11C10E1 n =20.58, transfection efficiency increases with the increase in the degree of polymerization. Of the first 5 PBAE with the highest transfection efficiency in vivo, 3/5 of the different blocking groups belong to group E1. Comparison of 11C10E1, 11C12E1/11C14E1, 11C16E1 and 11C18E1 in terms of carbon chain length in groups n =9-12 or 17-26 led to the conclusion that the closer the carbon chain length was to 16, the better the transfection effect still applies.
Example 4 evaluation of toxicity of FNP prescription in mice
Good formulations require not only high transfection efficiency but also good biocompatibility. For this reason, the in vivo toxicity of the first 5 PBAE was next evaluated using a higher mRNA dose (0.6 mg/kg).
Male C57BL/6 mice weighing 15-20g were randomly grouped into 4 mice per group. The prepared FNP preparation is administrated via tail vein according to luciferase mRNA dosage of 0.6 mg/kg. The positive control group was injected intraperitoneally with lipopolysaccharide (5 mg/kg), and the negative control group was injected intravenously with PBS. After 48h, whole blood was collected and serum was centrifuged. Mouse organs (heart, liver, spleen, lung, kidney) were sectioned and stained with H & E. The liver (AST, ALT) and kidney (BUN, CREA) function was then tested with the kit.
FNP with the first 5 PBAE did not change renal and hepatic function, as shown in fig. 7, and fig. 8 shows no evidence of histologic adverse damage. The above results indicate that FNP has good tolerance.
Example 5
FNP nanoparticles were prepared for cryo-electron microscopy imaging, and after negative staining with 2% uranyl acetate, FNP was imaged with Tecnai Spirit transmission electron microscopy (FEI, hillsboro, OR). Cryo-electron microscopy results (fig. 9) show that FNP composed of 11C18E1 n =23.64 has a rosette structure and a particle size of about 70nm.
mRNA encapsulation efficiency by different FNPs was evaluated using a modified Quant-iT RiboGreen RNA assay (Invitrogen) kit.
FNP formulations for different PBAE materials were prepared using the method of the above example, and after preparation the FNP was characterized by diluting the FNP 40 fold in DPBS solution and measuring its size, polydispersity (PDI) and Zeta potential using a NanoZS Zetasizer (Malvern).
As shown in FIGS. 10 and 11, the most effective sets of PBAE had the smallest particle size, the smallest PDI, and the encapsulation efficiency was nearly 80%, and the four most effective FNPs all had surface zeta potentials near +5mV, which were neither too high nor too low.
Example 6 evaluation of hemolysis of different FNPs
Male C57BL/6 mice weighing 15-20g were selected, whole blood was taken, and blood cells were separated by centrifugation (10000g, 5min). Then, the blood cells were resuspended in PBS solutions of pH7.4 and Ph5.0, different FNP materials were added thereto, incubated at 37 ℃ for 30min, centrifuged at 10000g for 5min, the supernatant was collected and its absorbance at 540nm was measured, and the group without FNP nanoparticles was used as a negative control and Triton X-100 solution (1 wt.%) was used as a positive control.
The membrane rupture activity of FNP was evaluated by a hemolysis assay, and as shown in fig. 12, PBAE with good transfection efficiency showed higher hemolysis ability at ph5.0, indicating higher escape ability in lysosome acidic environment.
Example 7 evaluation of FNP uptake and escape in 293T cells
293T cells were seeded in 8-well glass plates (CellVis, mountain View, CA, cat. C8-1.5H-N). When the cell density reached 60-70%, 375ng cy5 marker mRNA-coated FNP was added to each well, and after 4 hours, the image was taken on a Nikon confocal microscope (Ti-E + A1R SI) with a 60-fold oil immersion objective. The nucleus and lysosome were labeled with DAPI and LysoTracker Red DND-99, respectively.
Lysosomes and mRNA were labeled with LysoTracker and cy5, respectively, mRNA uptake was determined by semiquantitative analysis of cy5 signals in individual cells by imageJ, and endosomal escape capacity was semiquantitatively analyzed by the dispersion of cy5 and LysoTracker.
Cellular uptake assays were performed using ImageJ (FIJI). The area where the single cell is located is selected in the image, and the FNP uptake of the single cell is indicated by calculating the fluorescence intensity of cy5 in the area. Coloc 2 plug-in from ImageJ (FIJI) was used for co-localization analysis to indicate lysosome escape by calculating the pearson correlation coefficient for the far-red (cy 5-mRNA) channel and the red (endosomal marker). Wherein, the closer the pierce correlation coefficient is to 0, the lower the correlation between two channels, which indicates the higher the efficiency of lysosome escape.
As shown in FIGS. 13-15, FNP with good transfection effect on cells and in vivo has high uptake effect and good lysosome escape ability.
Example 8 evaluation of stability of FNP nanoparticles from the Change in transfection ability of cells over time
FNP was prepared in the same manner as in the above example, sucrose was added to a final concentration of 10% (w/v) after completion of the preparation, and freeze-dried for 12 hours using a freeze-dryer (Labconco, USA) after prefreezing at-80 deg.C until the water was completely evaporated. The freeze-dried FNP was stored at 4 deg.C and the original volume of ddH was used at selected time points 2 The O re-dissolves. FNP containing 150ng nucleic acid was mixed with 100uL DMEM (High glucose) Complete Medium (10% FBS), added to 293T cells at a density of 80% to 90% and replaced with the original Medium. Luciferase expression was measured after 24 hours.
FIGS. 16-17 show that the transfection effect of the conventional formulations for C12-200 and Dlin-MC3 is significantly reduced after lyophilization and reconstitution, and that the transfection effect of the nanoparticles made positively surface-charged by adding 50% DOTAP to the formulations for C12-200 and Dlin-MC3 can be maintained substantially unchanged after lyophilization, while the incorporation of 50% into the formulations for Dlin-MC3 is 50: after the two types of nanoparticles with negative electricity on the surfaces of the PA are freeze-dried, the transfection effect is obviously reduced; whether the PBAE alone prescription can maintain transfection efficiency after lyophilization by facilitating encapsulation of nucleic acids. This indicates that the addition of both DOTAP and PBAE can effectively increase the lyophilization stability of the nanoparticles.
Figure 18 shows FNP nanoparticles after lyophilization and long term storage at 4 ℃ and their cell transfection efficiency measured at selected time points, with the first 4 PBAEs being more stable and the 5 th ones being less stable, with the ability to transfect cells decreasing over time. Compared with the freeze-dried group, the FNP transfection effect decreased rapidly when the cells were stored at 4 ℃ without freeze-drying.
In addition, changes in FNP particle size and polydispersity index (PDI) were measured at selected time points for long term storage after lyophilization using Zetasizer Nano-ZS (Malvern, UK) Dynamic Light Scattering (DLS). Measurements for each sample were repeated 3 times.
The results show that: the FNP nanoparticles also remained essentially stable in particle size, PDI and zeta potential over time, and compared to the control group, FNP had good stability and could be kept stable for a long period of time after lyophilization, since the addition of cationic lipids to FNP increased its surface charge, resulting in increased repulsion between its particles, while the addition of polymer PBAE resulted in tighter encapsulation of nucleic acids, which could better withstand the lyophilization process (fig. 19).
Example 9 evaluation of efficiency of editing to deliver cre mRNA in Ai9 mice
To detect and quantify the cell type and proportion of FNP entering the lung, we used a transgenic mouse with a LoxP-flanked tdTom reporter (Ai 9), a transgenic mouse with a LoxP-flanked STOP sequence inserted at the Gt (ROSA) 26Sor locus to prevent transcription of the CAG promoter-driven red fluorescent protein (tdTomato). Ai9 mice express strong tdTomato fluorescence following Cre-mediated recombination. Cre mRNA (0.3 mg/kg) was loaded in FNP by tail vein injection, and tdTomato red fluorescent protein expression was detected by a Living body imager 48 hours later.
Example 10 evaluation of FNP distribution in various types of cells in the Lung
Ai9 mice were sacrificed by tail vein injection of FNP loaded with cre mRNA (0.3 mg/kg) and 48 hours later, dissected for 1mL Dispase (15U/mL) injected through trachea into lungs, after lungs auto-collapse, lungs were removed and placed in PBS solution, lung lobes were separated and tracheal tissue was removed, placed in Dispase (15U/mL) for digestion for 45min, then lung tissue was triturated with a pipette until no obvious tissue mass was seen, then 10ml DMEM 2 FBS + 1P/S +0.33U/mL DNaseI was added, vigorously blown with a pipetteThen placing the mixture in a shaking table at 37 ℃ to shake for 10min, then passing through cell filter membranes of 100 mu m,70 mu m and 40 mu m, centrifuging at 1200rpm for 5min to collect cells, adding 1mL of red blood cell lysate to resuspend and lyse the red blood cells, then centrifuging again to collect the cells, adding DMEM + 2-FBS + P/S to resuspend the cells and recovering at 37 ℃ for at least 20 min, counting by using a cell counting plate to ensure that the cell density is 10 7 and/mL. Cell staining was then performed using antibodies Pacific Blue (BioLegend, 157212) against mouse CD45, alexa Fluor 488 against mouse CD31 (BioLegend, 102414) and Alexa Fluor 647 against mouse CD326 (Ep-CAM) (BioLegend, 118212). Ghost Dye Red 780 (cell signaling, 18452S) was used to identify viable cells. Lung cells were analyzed by lsrforesa SORP.
The type and proportion of cells edited by Cre recombinase can be detected by delivering FNP encapsulating Cre mRNA. Cre mRNA was delivered with 5 most potent FNPs, and 2 days after administration, fluorescent tissue was evident in lung tissue, as shown in FIG. 20. We note that the FNP of 11C16E2 n =24.11 composition has a 6-9 times higher fluorescence signal in its lungs than the PBS group. In addition, tdTom-positive cells were easily seen under a tissue section fluorescence microscope, as shown in FIG. 21. To estimate the cell types that 11C16E2 can transmit (n = 24.11), we quantified the specific cell types delivered into the lung using flow cytometry after Cre mRNA delivery. 11C16E2 n =24.11fnp transfected about 22% of all endothelial cells, about 6% of epithelial cells and about 8% of immune cells, as shown in fig. 22.
If one wants to optimize this formulation, obtain a more surprising delivery efficiency, and lay the foundation for extending the FNP platform to more accurate cell targeting, one must study the mechanistic factors that determine the FNP lung targeting properties.
Example 11 evaluation of FNP distribution in mice
For lung targeting, nanoparticles first need to be specifically distributed to the lungs. Therefore, we wish to verify whether the FNPs are all distributed in the lungs. FNP consisting of 111c18E1 n-23.64 and cy5-EGFP mRNA is injected into a mouse through tail vein, and after 6h, the IVIS Lumina system and RT-qPCR are respectively used for detecting cy5 fluorescence distribution and the content of EGFP mRNA in each organ.
C57BL/6 mice weighing 15-20g were injected intravenously with FNP and PBS solutions encapsulating EGFP mRNA (0.3 mg/kg), respectively, 3 per group. After 6 hours, mice were sacrificed and organs removed and total RNA was extracted using Vazyme cat. Rc112-01. cDNA was synthesized by Vazyme Cat. R223-01. Then, the qPCR assay was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Cat. Q711-02). The RT-qPCR primers are GAPDH:5'-CGACTTCAACAGCAACTCCCACTCTTCC-3' (forward), primer 5'-TGGGTGGTCCAGGGTTTCTTACTCCTT-3' (reverse); EGFP:5'-CGACCACTACCAGCAGAACA-3' (forward) and EGFP primer 5'-TCTCGTTGGGGTCTTTGCTC-3' (reverse). PBS group is negative control group, GAPDH is endogenous control.
FIG. 23a shows that cy5 fluorescence signal is mainly distributed in lung, and only a small amount of fluorescence signal is scattered in liver, kidney and spleen (fluorescence signal in lung is about 2 times of that in liver, kidney and spleen). As shown in FIG. 23b, RT-qPCR results showed that cy5-EGFP mRNA was mainly distributed in the lung, which was much higher than other organs (lung fluorescence signals were at least 10 times higher than those of liver, kidney, spleen). The two experimental results show that the distribution ratio of mRNA in lung and other organs is different, the fluorescence imaging result shows that a small part of cy5 signals on EGFP mRNA are distributed in liver, kidney and spleen, but the content of mRNA in each organ is measured by RT-qPCR, but the content of non-lung organs is low, which is probably because of degradation and metabolism.
This explains to some extent why nanoparticles are specifically expressed only in the lung, but to really understand the mechanism, it is also necessary to understand why these nanoparticles are specifically expressed in the lung and what promotes their uptake by the lung.
Example 12 analysis of protein crown composition bound by FNP and verification of the Effect of the protein of interest on the transfection of Lung Primary cells
Traditional four-component LNPs can deliver functional RNA to hepatocytes by adsorbing ApoE in serum and binding to low density lipoprotein receptor (LDL-R) on hepatocytes. We therefore hypothesized that FNP is also a specific lung endothelial cell targeting by binding to proteins in serum.
FNP and mouse serum were incubated at 37 deg.C and simulatedFormation of protein corona in vivo, FNP bound to protein corona by centrifugation, and adding 6 times volume of cold acetone to the protein corona separated from FNP to remove lipid content and precipitate protein. 16000g centrifugation at 4 deg.C for 15min, and resuspension of the resulting protein mixture in 1M Urea/50 mM NH 4 HCO 3 In (5), 1M DTT was added to give a final DTT concentration of 5mM, and the mixture was incubated at 37 ℃ for 1 hour. 500mM IAM was then added to give a final IAM concentration of 10mM and incubated at room temperature for 45min. Trypsin was added to digest the protein. The resulting polypeptide mixture was resuspended in 0.1% formic acid and analyzed with Q active HF (ThermoFisher).
The composition of the formed protein corona was analyzed by proteomics as shown in figure 24 a. Vitronectin is the most abundant plasma protein in FNP compared to the nanoparticle formulation consisting of control Dlin-MC 3. The different functions of vitronectin compared to ApoE may play a role in lung target recognition forming FNP. Vitronectin promotes cell attachment, diffusion and migration of a variety of cell types. At least four known integrin receptors, including alpha, recognize vitronectin v β 3 ，α v β 5 ，α v β 1 And alpha Ⅱb β 3 . Integrin alpha v β 3 Is a major vitronectin receptor that binds to vitronectin in plasma. In fact, integrin alpha v β 3 It is found in many cells, such as endothelial cells, chondrocytes, fibroblasts, and blasts. Alpha (alpha) ("alpha") v β 1 Are distributed among a limited number of cell types, such as fibroblasts and certain tumor cell lines. Alpha is alpha v β 5 There is also distribution in fibroblasts and tumors. And alpha is Ⅱb β 3 Mainly distributed in platelets and megakaryocytes, and require activation to bind vitronectin. After the nanoparticles are injected through tail veins of mice, the nanoparticles return to the heart through systemic circulation, then rapidly enter pulmonary circulation to exchange alveolar capillary networks around gas alveoli, then are injected into the left atrium and the left ventricle to pass through pulmonary veins of all levels, finally flow through the aorta and a plurality of branches of arteries generated by the aorta, and convey blood to corresponding organs for material exchange.
Thus, in combination with the two main aspects, we envision that FNP, once in the blood, adsorbs vitronectin, rapidly circulates through the heart to the lungs, and is highly expressed in alpha with lung endothelial cells v β 3 Receptor interaction. Therefore, most of the nanoparticles will be intercepted in the lung, thereby greatly reducing the distribution of the nanoparticles in the liver and kidney.
To verify the hypothesis, FNP loaded with eGFP mRNA and different doses of vitronectin were incubated to transfect α separately v β 3 Receptor positive U87 cell line, mouse lung primary microvascular endothelial cells, and alpha v β 3 Receptor negative HepG2 cell lines, as shown in figure 24b, 24c. At α v β 3 In a receptor positive U87 cell line and primary mouse lung microvascular endothelial cells, the transfection efficiency is obviously improved along with the increase of vitronectin. And at alpha v β 3 In the receptor-negative HepG2 cell line, there was no significant increase in transfection efficiency with increasing vitronectin. Description of alpha v β 3 The receptor is a key contributing factor in mediating cell transfection of vitronectin-enriched FNP. In addition, we incubated FNP with different doses of bovine serum albumin for transfection of α v β 3 The transfection efficiency of the receptor positive U87 cell line is not obviously increased along with the increase of bovine serum albumin. This demonstrates vitronectin mediated alpha in FNP v β 3 Also plays a critical role in the transfection of receptor positive cells.
In summary, after FNP is introduced into the body, vitronectin in blood is rapidly enriched and passes through and alpha with the lung v β 3 Receptor interaction, mediating lung specific transfection.
Example 13 evaluation of properties after FNP Loading of pDNA
The effect of FNP loading pDNA was tested and FNP formulations were prepared in the same way as when mRNA was encapsulated.
Changes in FNP particle size and polydispersity index (PDI) were measured after lyophilization for long term storage using Zetasizer Nano-ZS (Malvern, UK) Dynamic Light Scattering (DLS). Measurements for each sample were repeated 3 times; the encapsulation of pDNA encapsulated by FNP was analyzed by agarose gel electrophoresis.
Particle size, PDI and Zeta potential of FNP-pDNA were measured using Dynamic Light Scattering (DLS), and figure 26a shows that FNP has no significant difference in nanoparticle size and potential for encapsulating pDNA and mRNA. FIG. 26b agarose gel electrophoresis results show that FNP can effectively encapsulate pDNA.
Transfection effects of FNP prescription to transfect luciferase pDNA in mice were evaluated. The nanoparticle preparation method was the same as in the third example. Then the luciferase is injected into a mouse body through tail vein, 30mg/mL of luciferin potassium salt is injected into the abdominal cavity according to the dose of 150mg/kg after six hours of administration, and the luciferase expression condition is detected about ten minutes after the injection.
Finally, the transfection effect of the FNP prescription for transfecting luciferase pDNA in a mouse is verified. FIG. 27 shows that lung-specific luciferase expression can be efficiently achieved at a plasmid dose of 0.3 mg/kg.
Example 14 evaluation of the Effect of transfection of FNP on 293T cells prepared with different PBAE materials at different prescribed ratios
FNP nanoparticles coated with EGFP pDNA were prepared by rapidly mixing (DOTAP + PBAE)/DOPE/Chol/DMG-PEG at a molar ratio of 50/10/38.5/1.5 in ethanol with an aqueous phase containing nucleic acids (100 mM sodium citrate buffer) at a volume ratio of 1:1. The prepared nanoparticles are directly added into a complete DMEM culture medium to transfect cells, and the expression level is observed under a fluorescence microscope after 24 hours. The molar ratio of DOTAP to PBAE is varied from 5/1 to 20/1 according to different types of PBAE, and the optimal proportion of each PBAE material is selected according to the transfection efficiency.
For in vitro cell transfection, fresh FNP was directly diluted in DMEM complete medium; for in vivo delivery, prepared FNP was dialyzed against 3500mwco dialysis membrane in PBS solution at 4 ℃ for 2h. FIG. 25 shows the effect of transfection of different PBAE materials on 293T cells under different formulation conditions.
Claims (11)
1. A polymeric (β -amino ester) characterized in that it comprises a compound of formula (I);
wherein:
R 1 independently a fatty chain with or without hydroxyl groups;
R 2 independently is a terminal monomer comprising a primary, secondary or tertiary amine;
r comprises a straight or branched C 1 -C 50 An alkylene chain which may further comprise one or more heteroatoms or one or more carbocyclic, heterocyclic or aromatic groups;
m is 1-15, n is 3-45, and x is 1-13.
2. The polymerized β -aminoester of claim 1, wherein:
the R is 1 Independently is
And/or the presence of a gas in the gas,
the R is 2 Independently is
R is independently
3. A polymeric (β -amino ester) according to claim 1 or 2, prepared by a process comprising: r is to be 1 、R 2 R and alkylamine in admixtureTogether, a michael addition reaction is performed.
4. A five-membered lipid nanoparticle, starting from a cationic lipid, a poly (β -aminoester) according to any one of claims 1 to 3, a sterol, a phospholipid, a PEG lipid and a nucleic acid molecule.
5. The five-membered lipid nanoparticle according to claim 4, wherein the molar ratio of said cationic lipid to said PBAE is (2.5-40): 1;
and/or the relative amounts of the mass of the cationic lipid and the PBAE to the nucleic acid molecule are (10-80): 1;
and/or the molar ratio of the phospholipid, the sterol and the PEG lipid is phospholipid: sterol = (0.2-1): 1; phospholipid: PEG lipid = (5-20): 1;
and/or, the ratio of n to m is 7:3;
and/or, x is 3 to 11.
6. The five-membered lipid nanoparticle according to claim 4, wherein said cationic lipid is selected from one or more of DOTAP, DOTMA, DODAB, DDAB, and DMRIE; preferably DOTAP;
and/or, the sterol is selected from one or more of cholesterol, campesterol, algal sterol and carrot sterol; preferably cholesterol;
and/or the phospholipid is selected from one or more of DOPE, DOPC, DOPS, POPS, DOPG, DOPI, DSPC, HSPC, eggPC, DPPC, POPC, POPE and DLPC; preferably DOPE;
and/or, the PEG lipid is selected from one or more of DMG-PEG2000, DMG-PEG5000, DSPE-PEG2000 and DSPE-PEG 5000;
and/or, the nucleic acid molecule is RNA and/or DNA.
7. The five-membered lipid nanoparticle according to claim 4, wherein the preparation method comprises:
dissolving a cationic lipid, a poly (β -aminoester) according to any of claims 1 to 3, a sterol, a phospholipid and a PEG lipid into an ethanol phase, and dissolving a nucleic acid molecule into an aqueous phase;
mixing the two phases to obtain the five-membered lipid nanoparticles;
the two phases are preferably mixed in a volume ratio of ethanol to water of 1:1.
8. The five-membered lipid nanoparticle according to any one of claims 4 to 7, wherein the starting materials comprise DOTAP, PBAE, cholesterol, DOPE, and PEG lipids in a molar ratio (DOTAP + PBAE) of DOPE: cholesterol: DMG-PEG = 50.
9. A pharmaceutical composition or vaccine comprising the five-membered lipid nanoparticle of any one of claims 4-8 and a pharmaceutically acceptable carrier.
10. Use of a polymerized (β -aminoester) according to any of claims 1 to 3 for the preparation of a pentameric lipid nanoparticle.
11. A lyophilized formulation comprising the five-membered lipid nanoparticle according to any one of claims 4 to 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210114678.7A CN115304756B (en) | 2022-01-30 | 2022-01-30 | Five-membered lipid nanoparticle as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210114678.7A CN115304756B (en) | 2022-01-30 | 2022-01-30 | Five-membered lipid nanoparticle as well as preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115304756A true CN115304756A (en) | 2022-11-08 |
| CN115304756B CN115304756B (en) | 2023-05-09 |
Family
ID=83855169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210114678.7A Active CN115304756B (en) | 2022-01-30 | 2022-01-30 | Five-membered lipid nanoparticle as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115304756B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115925563A (en) * | 2023-02-28 | 2023-04-07 | 清华大学 | Lipid molecule for targeted lung delivery of nucleic acid and preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106659803A (en) * | 2014-04-23 | 2017-05-10 | 摩登纳特斯有限公司 | Nucleic acid vaccines |
| CN109476718A (en) * | 2016-05-18 | 2019-03-15 | 莫得纳特斯公司 | The combination of MRNA and application thereof of encoding immune adjusting polypeptide |
| CN110582302A (en) * | 2016-12-14 | 2019-12-17 | 利甘达尔股份有限公司 | Compositions and methods for nucleic acid and/or protein payload delivery |
| CN111971066A (en) * | 2018-01-18 | 2020-11-20 | 伊泽阿恩埃免疫疗法股份有限公司 | Lipid nanoparticles |
| CN112143731A (en) * | 2020-09-14 | 2020-12-29 | 广州瑞风生物科技有限公司 | gRNA for targeted destruction of SARS-CoV-2 virus genome and its application |
| US20210032192A1 (en) * | 2019-07-30 | 2021-02-04 | Factor Bioscience Inc. | Cationic lipids and transfection methods |
| JP2021525240A (en) * | 2018-05-24 | 2021-09-24 | トランスレイト バイオ， インコーポレイテッド | Thioester cationic lipid |
-
2022
- 2022-01-30 CN CN202210114678.7A patent/CN115304756B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106659803A (en) * | 2014-04-23 | 2017-05-10 | 摩登纳特斯有限公司 | Nucleic acid vaccines |
| CN109476718A (en) * | 2016-05-18 | 2019-03-15 | 莫得纳特斯公司 | The combination of MRNA and application thereof of encoding immune adjusting polypeptide |
| CN110582302A (en) * | 2016-12-14 | 2019-12-17 | 利甘达尔股份有限公司 | Compositions and methods for nucleic acid and/or protein payload delivery |
| CN111971066A (en) * | 2018-01-18 | 2020-11-20 | 伊泽阿恩埃免疫疗法股份有限公司 | Lipid nanoparticles |
| JP2021525240A (en) * | 2018-05-24 | 2021-09-24 | トランスレイト バイオ， インコーポレイテッド | Thioester cationic lipid |
| US20210032192A1 (en) * | 2019-07-30 | 2021-02-04 | Factor Bioscience Inc. | Cationic lipids and transfection methods |
| CN112143731A (en) * | 2020-09-14 | 2020-12-29 | 广州瑞风生物科技有限公司 | gRNA for targeted destruction of SARS-CoV-2 virus genome and its application |
Non-Patent Citations (1)
| Title |
|---|
| 许从飞;王均;: "非编码RNA药物的研究进展", 生命科学 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115925563A (en) * | 2023-02-28 | 2023-04-07 | 清华大学 | Lipid molecule for targeted lung delivery of nucleic acid and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115304756B (en) | 2023-05-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Intracellular delivery and biodistribution study of CRISPR/Cas9 ribonucleoprotein loaded bioreducible lipidoid nanoparticles | |
| Ko et al. | Self-assembling micelle-like nanoparticles based on phospholipid–polyethyleneimine conjugates for systemic gene delivery | |
| de Wolf et al. | Effect of cationic carriers on the pharmacokinetics and tumor localization of nucleic acids after intravenous administration | |
| Karlsson et al. | Engineered nanoparticles for systemic siRNA delivery to malignant brain tumours | |
| Grosse et al. | Tumor‐specific gene transfer with receptor‐mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers | |
| Li et al. | Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy | |
| Li et al. | Non-viral strategies for delivering genome editing enzymes | |
| JP2011503070A (en) | Self-assembled micelle-like nanoparticles for systemic gene delivery | |
| US9730893B2 (en) | Lipid assemblies comprising anionic lysolipids and use thereof | |
| US10113151B2 (en) | Composition of viral vectors in lecithin liposomes, preparation method and treatment methods | |
| Zhang et al. | Ligand-installed anti-VEGF genomic nanocarriers for effective gene therapy of primary and metastatic tumors | |
| US20150297749A1 (en) | Low-density lipoprotein analogue nanoparticles, and composition comprising same for targeted diagnosis and treatment of liver | |
| CN115304756B (en) | Five-membered lipid nanoparticle as well as preparation method and application thereof | |
| JP2010059064A (en) | Drug delivery complex | |
| Distasio et al. | Design and development of Branched Poly (ß-aminoester) nanoparticles for Interleukin-10 gene delivery in a mouse model of atherosclerosis | |
| Zhuang et al. | Delivery systems of therapeutic nucleic acids for the treatment of acute lung injury/acute respiratory distress syndrome | |
| CN111450061A (en) | Hybrid mesenchymal stem cell exosome drug delivery system and preparation method and application thereof | |
| Zhang et al. | Combined self-assembled hendeca-arginine nanocarriers for effective targeted gene delivery to bladder cancer | |
| JP5787323B2 (en) | Lipid membrane structure | |
| AU2021351517A1 (en) | Proteolipid vesicles formulated with fusion associated small transmembrane proteins | |
| US20230320994A1 (en) | Functional ionizable phospholipids | |
| EP4346781A1 (en) | Dna vector delivery using lipid nanoparticles | |
| CN114540416A (en) | Expression vector, lipid nanoparticle, antitumor drug, preparation method and application thereof | |
| CN114848831A (en) | Coated nano preparation and preparation method and application of carrier thereof | |
| CN118079009A (en) | Lung-targeting polymer-lipid composition, nucleic acid delivery composition, preparation method and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |