CN115926134B - Cationic polyester and preparation method and application thereof - Google Patents
Cationic polyester and preparation method and application thereof Download PDFInfo
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- CN115926134B CN115926134B CN202211419916.1A CN202211419916A CN115926134B CN 115926134 B CN115926134 B CN 115926134B CN 202211419916 A CN202211419916 A CN 202211419916A CN 115926134 B CN115926134 B CN 115926134B
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- 125000002091 cationic group Chemical group 0.000 title claims abstract description 87
- 229920000728 polyester Polymers 0.000 title claims abstract description 86
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 239000000178 monomer Substances 0.000 claims abstract description 114
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 61
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 61
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 61
- 238000001890 transfection Methods 0.000 claims abstract description 55
- 229920000587 hyperbranched polymer Polymers 0.000 claims abstract description 48
- 238000012986 modification Methods 0.000 claims abstract description 48
- 230000004048 modification Effects 0.000 claims abstract description 48
- 239000003814 drug Substances 0.000 claims abstract description 28
- JNGZXGGOCLZBFB-IVCQMTBJSA-N compound E Chemical compound N([C@@H](C)C(=O)N[C@@H]1C(N(C)C2=CC=CC=C2C(C=2C=CC=CC=2)=N1)=O)C(=O)CC1=CC(F)=CC(F)=C1 JNGZXGGOCLZBFB-IVCQMTBJSA-N 0.000 claims abstract description 27
- 150000001875 compounds Chemical class 0.000 claims abstract description 24
- 238000012377 drug delivery Methods 0.000 claims abstract description 15
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 15
- -1 cyclic lactone Chemical class 0.000 claims abstract description 13
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 13
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 11
- 150000003512 tertiary amines Chemical class 0.000 claims abstract description 9
- 230000000379 polymerizing effect Effects 0.000 claims abstract description 7
- 150000007524 organic acids Chemical class 0.000 claims abstract description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract 2
- 108020004999 messenger RNA Proteins 0.000 claims description 85
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 45
- 239000000463 material Substances 0.000 claims description 26
- 239000002245 particle Substances 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 24
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 24
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 22
- 108020004414 DNA Proteins 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 21
- 239000007822 coupling agent Substances 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 13
- 238000006116 polymerization reaction Methods 0.000 claims description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 11
- CXMXRPHRNRROMY-UHFFFAOYSA-N sebacic acid Chemical compound OC(=O)CCCCCCCCC(O)=O CXMXRPHRNRROMY-UHFFFAOYSA-N 0.000 claims description 11
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- 108090001060 Lipase Proteins 0.000 claims description 8
- 239000004367 Lipase Substances 0.000 claims description 8
- 102000004882 Lipase Human genes 0.000 claims description 8
- 239000003054 catalyst Substances 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 claims description 8
- 235000019421 lipase Nutrition 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 7
- 150000003335 secondary amines Chemical class 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- FKUPPRZPSYCDRS-UHFFFAOYSA-N Cyclopentadecanolide Chemical compound O=C1CCCCCCCCCCCCCCO1 FKUPPRZPSYCDRS-UHFFFAOYSA-N 0.000 claims description 6
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Chemical compound C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 150000002500 ions Chemical class 0.000 claims description 6
- CRVGTESFCCXCTH-UHFFFAOYSA-N methyl diethanolamine Chemical compound OCCN(C)CCO CRVGTESFCCXCTH-UHFFFAOYSA-N 0.000 claims description 6
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 4
- LOKPJYNMYCVCRM-UHFFFAOYSA-N 16-Hexadecanolide Chemical compound O=C1CCCCCCCCCCCCCCCO1 LOKPJYNMYCVCRM-UHFFFAOYSA-N 0.000 claims description 4
- 108700011259 MicroRNAs Proteins 0.000 claims description 4
- 108091029810 SaRNA Proteins 0.000 claims description 4
- 108020004459 Small interfering RNA Proteins 0.000 claims description 4
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 claims description 4
- 239000012298 atmosphere Substances 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 150000001718 carbodiimides Chemical class 0.000 claims description 4
- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
- 239000012295 chemical reaction liquid Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 125000000623 heterocyclic group Chemical group 0.000 claims description 4
- 239000002679 microRNA Substances 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 229940078677 sarna Drugs 0.000 claims description 4
- KQTIIICEAUMSDG-UHFFFAOYSA-N tricarballylic acid Chemical compound OC(=O)CC(C(O)=O)CC(O)=O KQTIIICEAUMSDG-UHFFFAOYSA-N 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 2
- VQNDBXJTIJKJPV-UHFFFAOYSA-N 2h-triazolo[4,5-b]pyridine Chemical compound C1=CC=NC2=NNN=C21 VQNDBXJTIJKJPV-UHFFFAOYSA-N 0.000 claims description 2
- WXSRRNYRGPTTNP-UHFFFAOYSA-N 3-(hydroxymethyl)pentane-1,5-diol Chemical compound OCCC(CO)CCO WXSRRNYRGPTTNP-UHFFFAOYSA-N 0.000 claims description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 2
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 2
- AKNUHUCEWALCOI-UHFFFAOYSA-N N-ethyldiethanolamine Chemical compound OCCN(CC)CCO AKNUHUCEWALCOI-UHFFFAOYSA-N 0.000 claims description 2
- ZJCCRDAZUWHFQH-UHFFFAOYSA-N Trimethylolpropane Chemical compound CCC(CO)(CO)CO ZJCCRDAZUWHFQH-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 230000009471 action Effects 0.000 claims description 2
- 235000011037 adipic acid Nutrition 0.000 claims description 2
- 239000001361 adipic acid Substances 0.000 claims description 2
- 125000003545 alkoxy group Chemical group 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 125000003118 aryl group Chemical group 0.000 claims description 2
- RROBIDXNTUAHFW-UHFFFAOYSA-N benzotriazol-1-yloxy-tris(dimethylamino)phosphanium Chemical compound C1=CC=C2N(O[P+](N(C)C)(N(C)C)N(C)C)N=NC2=C1 RROBIDXNTUAHFW-UHFFFAOYSA-N 0.000 claims description 2
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 claims description 2
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 229940094933 n-dodecane Drugs 0.000 claims description 2
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 claims 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims 2
- 239000011574 phosphorus Substances 0.000 claims 2
- WAUVRUBPGUOVEI-UHFFFAOYSA-N 1-n,1-n,2-n,2-n-tetrahydroxybutane-1,2-diamine Chemical compound CCC(N(O)O)CN(O)O WAUVRUBPGUOVEI-UHFFFAOYSA-N 0.000 claims 1
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 14
- 230000003013 cytotoxicity Effects 0.000 abstract description 14
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 125000000467 secondary amino group Chemical class [H]N([*:1])[*:2] 0.000 abstract 2
- 229920000642 polymer Polymers 0.000 description 38
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 32
- 238000012360 testing method Methods 0.000 description 28
- 208000035823 Non-specific autoimmune cerebellar ataxia without characteristic antibodies Diseases 0.000 description 27
- CDBAMNGURPMUTG-UHFFFAOYSA-N 4-[2-(4-hydroxycyclohexyl)propan-2-yl]cyclohexan-1-ol Chemical compound C1CC(O)CCC1C(C)(C)C1CCC(O)CC1 CDBAMNGURPMUTG-UHFFFAOYSA-N 0.000 description 15
- 230000000694 effects Effects 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 11
- 238000001727 in vivo Methods 0.000 description 10
- 230000002685 pulmonary effect Effects 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000002715 modification method Methods 0.000 description 5
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000012096 transfection reagent Substances 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000005022 packaging material Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
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- 231100000419 toxicity Toxicity 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229910052786 argon Inorganic materials 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 150000002431 hydrogen Chemical group 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
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- 108090000331 Firefly luciferases Proteins 0.000 description 2
- 241000607734 Yersinia <bacteria> Species 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001476 gene delivery Methods 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 238000012637 gene transfection Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 150000003141 primary amines Chemical group 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- HLHSUNWAPXINQU-GQCTYLIASA-N (E)-3-(3,4-dihydroxyphenyl)-N-prop-2-ynylprop-2-enamide Chemical compound OC=1C=C(C=CC=1O)/C=C/C(=O)NCC#C HLHSUNWAPXINQU-GQCTYLIASA-N 0.000 description 1
- PCGISRHGYLRXSR-UHFFFAOYSA-N 4-hydroxy-7-[(5-hydroxy-7-sulfonaphthalen-2-yl)carbamoylamino]naphthalene-2-sulfonic acid Chemical compound OC1=CC(S(O)(=O)=O)=CC2=CC(NC(=O)NC=3C=C4C=C(C=C(C4=CC=3)O)S(O)(=O)=O)=CC=C21 PCGISRHGYLRXSR-UHFFFAOYSA-N 0.000 description 1
- 102100021851 Calbindin Human genes 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 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 1
- 101000898082 Homo sapiens Calbindin Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
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- XYFCBTPGUUZFHI-UHFFFAOYSA-O phosphonium Chemical compound [PH4+] XYFCBTPGUUZFHI-UHFFFAOYSA-O 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000771 poly (alkylcyanoacrylate) Polymers 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
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- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Abstract
The application discloses cationic polyester, and a preparation method and application thereof. The cationic polyester is a hyperbranched polymer formed by polymerizing a monomer P, a monomer S and a monomer M or two monomers P and S with a monomer T, and the end group of the hyperbranched polymer is modified by a group E; monomer P is cyclic lactone; the monomer S is an organic acid with two or more carboxyl groups at the end group; the monomer M is a compound containing two hydroxyl groups and one secondary amine or tertiary amine; the monomer T is a compound containing three or more hydroxyl groups; the end group modifying compound E which provides modification of the group E is a compound containing at least one primary, secondary or tertiary amine group. The cationic polyester can remarkably improve the nucleic acid transfection efficiency and has lower cytotoxicity when being used for nucleic acid drug delivery, and can be used for clinic. In addition, the cationic polyester of the application has low preparation cost, environmental protection and no pollution.
Description
Technical Field
The application relates to the technical field of nucleic acid delivery materials, in particular to cationic polyester, a preparation method and application thereof.
Background
Gene therapy based on nucleic acid drug delivery is believed to lead to the third industrial revolution of biopharmaceuticals. Compared with other technologies such as DNA or virus vectors, the mRNA technology has the advantages of high efficiency, high safety, shorter production period, lower production cost and the like. mRNA technology has been well-known in the field of new crown vaccines, and is expected to bring new methods for various fields such as cancer and immune diseases. However, the application of this technology has been limited due to instability of mRNA molecules, low in vivo delivery efficiency, and the like. To achieve widespread use of mRNA technology, a suitable delivery vehicle is required to deliver it into the body to have a practical effect. The delivery technology that is currently commercially successful is mainly the Lipid Nanoparticle (LNP) technology based on ionizable cationic lipids used by companies such as Moderna and Biotech. Although LNP has been widely used for new coronal vaccines; however, the relatively low efficiency and significant side effects of LNP have the disadvantage of greatly limiting their range of application. How to develop new efficient and non-toxic delivery systems remains a bottleneck to inhibit the development of mRNA technology.
A biodegradable cationic polyester useful for DNA and mRNA delivery and a method for its preparation have previously been reported at the university of us. The preparation method is based on in-vitro synthesis biology technology, and the linear polymer is obtained by catalyzing polymerization of 3 monomers through immobilized lipase, and can be directly used or subjected to terminal group modification, so that the biodegradable cationic polyester for delivering DNA and mRNA can be obtained. Cationic polyesters at the university of Yersinia have higher transfection efficiency and lower toxicity compared to commercial reagents. However, the cationic polyester is still not efficient enough to be transfected and needs to be dissolved with a toxic organic solvent, making it difficult to be used in practical clinic.
Thus, it remains a major and difficult task in the art to develop nucleic acid delivery materials that are more efficient, non-toxic or less toxic to transfection.
Disclosure of Invention
The application aims to provide a novel cationic polyester, and a preparation method and application thereof.
In order to achieve the above purpose, the present application adopts the following technical scheme:
the first aspect of the application discloses a cationic polyester, which is a hyperbranched polymer formed by polymerizing a monomer P, a monomer S and a monomer M or two monomers P and S with a monomer T, wherein the end group of the hyperbranched polymer is modified by a group E; monomer P is cyclic lactone; the monomer S is an organic acid with two or more carboxyl groups at the end group; the monomer M is a compound containing two hydroxyl groups and one secondary amine or tertiary amine; the monomer T is a compound containing three or more hydroxyl groups; the end group modifying compound E which provides modification of the group E is a compound containing at least one primary, secondary or tertiary amine group.
It should be noted that, the modification of the group E by polymerizing P, S, M three monomers to obtain a linear polymer and a terminal group is a technology already reported by the university of Yersinia; the application is based on research, and found that a new polymer with hyperbranched structure, namely the new cationic polyester of the application, can be obtained by adding a monomer T into three monomers or directly adopting the monomer T to replace the monomer M. In addition, the research shows that when the cationic polyester is used for nucleic acid delivery, the gene transfection efficiency can be remarkably improved, the cytotoxicity is lower, and the clinical application can be better satisfied.
In one embodiment of the application, the monomer P is a cyclic lactone having a fatty chain length of 6 to 35.
In one embodiment of the present application, the monomer P is selected from at least one of, but not limited to, cyclohexanolactone, cyclododecanolactone, cyclopentadecanolide, and cyclohexadecanolide.
In one implementation of the application, monomer S is an organic acid having a carbon chain length of 3 to 18.
In one implementation of the application, the monomer S is selected from, but not limited to, at least one of adipic acid, sebacic acid, and 1,2, 3-propane tricarboxylic acid.
In one embodiment of the application, the monomers M are compounds having a carbon chain length of 4 to 36, comprising two hydroxyl groups, and one secondary or tertiary amine.
In one implementation of the application, the monomer M is selected from, but not limited to, at least one of diethanolamine, methyldiethanolamine and ethyldiethanolamine.
In one implementation of the application, monomer T is a compound having three or more hydroxyl groups with a carbon chain length of 4 to 54.
In one embodiment of the present application, the monomer T is selected from at least one of, but not limited to, trimethylol propane, 3- (hydroxymethyl) -1, 5-pentanediol, triethanolamine, N' -tetraethyleneglycol diamine.
In one implementation of the application, the end group modifying compound E providing modification of the group E is at least one of E1 to E26.
Preferably, the end group modifying compound E is at least one of E1 to E10, E12, E14 to E21, E25, E26.
More preferably, the end group modifying compound E is at least one of E2, E4, E9, E10, E12, E14, E15, E25, E26.
It should be noted that the cationic polyesters with end group modification by adopting the end group modification compounds E1 to E26 have better transfection efficiency and lower cytotoxicity; in particular, the transfection efficiency of the cationic polyesters modified by the end groups of E2, E4, E9, E10, E12, E14, E15, E25 and E26 is obviously better than that of other cationic polyesters modified by the end groups, and is also obviously better than that of lipoMM which is the most efficient commercial transfection reagent currently.
In one implementation of the application, the cationic polyester is a hyperbranched polymer of the structure shown in formula one;
one (I)
Wherein x, y, z are independent integers from 1 to 200,
n is an integer of 0 to 200,
j. k is an integer of 0 to 30,
l, m, o, p, q is an independent integer from 1 to 20,
R x is hydrogen, or a substituted or unsubstituted alkyl group having 1 to 18 carbon atoms, or a substituted or unsubstituted aryl group having at least 1 benzene ring, or a substituted or unsubstituted heterocyclic group having at least 1 heterocyclic ring, or a substituted or unsubstituted alkoxy group having 1 to 18 carbon atoms and at least 1 oxygen atom;
J is hydrogen, R 1 Modification of the group E provided for the end group-modifying compound E, in which case there is no R 2 The method comprises the steps of carrying out a first treatment on the surface of the Alternatively, J is carbonyl, in which case R 1 And R is 2 Both are the group E modifications provided by the end group modifying compound E;
in the formula I, the broken line represents a branch structure, the first monomer connected with the branch structure is P or S, and other monomers are connected subsequently to form a hyperbranched structure.
Wherein, the connection of other monomers means, for example, the subsequent connection of monomers according to the sequence of formula one, the formation of a branched structure on the basis of the branched structure, and the circulation in this way, the formation of a dendritic-like hyperbranched structure.
R of the present application 1 And/or R 2 And when the end group modification compound E modifies the end group E of the hyperbranched polymer, the end group modification compound E reduces one hydrogen to form a group E which is combined with the end group of the hyperbranched polymer. When N, N' -Carbonyl Diimidazole (CDI) is adopted as a coupling agent, end group modification of a group E is formed at two ends of the hyperbranched polymer shown in the formula I; in this case, in order to attach the group E to the hyperbranched polymer, at the end group R 2 CDI will provide one carbonyl group for linking group E and the hyperbranched polymer; r is R 1 The end group modifying compound E, which is still in place, will reduce by one hydrogen, forming a group E, which is directly linked to the hyperbranched polymer.
It should also be noted thatWhen only monomer P, S is polymerized with monomer T to form a hyperbranched polymer, i.e., n is 0, where J is a carbonyl group, one end of the carbonyl group is attached to R 2 The other end is connected with the hydroxyl of the monomer T, and the group E forming the end group is modified. It will be appreciated that in this case the structure of formula one described will be adapted, i.e. monomer T is terminally attached to carbonyl J.
In one implementation of the application, the cationic polyester has a number average molecular weight of 1 to 30k; preferably 2-20k.
The second aspect of the application discloses a method for preparing the cationic polyester of the application, comprising the steps of adding monomers and a catalyst into a solvent, sequentially carrying out a first-stage polymerization reaction and a second-stage polymerization reaction in an inert atmosphere, and removing the catalyst after the reaction is finished to obtain a hyperbranched polymer; then, under the action of a coupling agent, adopting a terminal group modification compound E to carry out terminal group modification on the hyperbranched polymer to obtain cationic polyester with terminal groups modified by the group E; wherein, the conditions of the first stage polymerization reaction are that the temperature is 85-95 ℃, the reaction vacuum degree is 50-1000 mbar, and the reaction time is 12-24 hours; the conditions of the second stage polymerization reaction are that the temperature is 85-95 ℃, the reaction vacuum degree is 1-30mbar, and the reaction time is 12-72 h.
In one implementation mode of the application, the molar ratio of the monomer P to the monomer S is 0.1:10-4:1, the molar ratio of the monomer M to the monomer S is 0:10-20:10, and the molar ratio of the monomer T to the monomer S is 0.1:10-20:10.
In one implementation of the application, the catalyst is an immobilized lipase.
In one implementation of the application, the amount of immobilized lipase is 3-50wt% of the total mass of each monomer.
In one implementation of the application, the solvent is at least one of diphenyl ether, n-dodecane, 1-butyl-3-methylimidazole hexafluorophosphate, dimethylacetamide and phthalic ether.
In one implementation of the application, the solvent is used in an amount of 100 to 500wt% of the total mass of the monomers.
In one implementation mode of the application, the catalyst is removed, specifically comprising the steps of filtering the reaction liquid by a filtering device after the reaction is finished, and collecting filtrate; the preparation method comprises the steps of adding n-hexane into filtrate to precipitate the hyperbranched polymer, centrifuging, removing supernatant, adding dichloromethane into precipitate to dissolve, adding n-hexane to precipitate the hyperbranched polymer, centrifuging, removing supernatant, and repeatedly dissolving with dichloromethane, precipitating with n-hexane, and centrifuging for at least 2 times; finally, the precipitate is dried, and the hyperbranched polymer is obtained.
In one implementation of the present application, the coupling agent is at least one of N, N' -carbonyldiimidazole, carbodiimide, phosphorous plus ion species and urea plus ion species.
In one implementation of the application, the carbodiimides include, but are not limited to, diisopropylcarbodiimide, dicyclohexylcarbodiimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
In one implementation of the application, the phosphorus cation species include, but are not limited to, benzotriazol-1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate; urea cations include, but are not limited to, 2- (7-azabenzotriazol) -N, N '-tetramethylurea hexafluorophosphate and O-benzotriazol-N, N' -tetramethylurea tetrafluoroboric acid.
In one implementation mode of the application, the molar ratio of the coupling agent to the hyperbranched polymer is 2:1-50:1; preferably, the molar ratio of the coupling agent to the hyperbranched polymer is 4:1 to 15:1; in addition, the molar ratio of the coupling agent to the end group modification compound E is 5:1 to 1:10.
In one implementation mode of the application, the end group modification compound E is adopted to carry out end group modification on the hyperbranched polymer, and specifically comprises the steps of dissolving the hyperbranched polymer in dichloromethane, adding a coupling agent, stirring for at least 10 hours at room temperature in an inert atmosphere, concentrating a reaction solution, adding diethyl ether with the volume of at least 3 times, centrifuging, removing sediment, and obtaining supernatant; removing the solvent in the supernatant, adding the dried product into dichloromethane for dissolution, adding the end group modification compound E under stirring, and reacting for at least 10 hours at room temperature to obtain the hyperbranched polymer with the end group modified by the group E, namely the cationic polyester.
In one implementation mode of the application, the preparation method of the application also comprises the steps of adding the equal volume of deionized water into the reaction liquid after the end group modification compound E is added to react at room temperature, and removing an upper water phase after vortex and centrifugal layering; repeatedly adding equal volume of deionized water, swirling, centrifuging for layering, and removing upper water phase for at least 3 times; and finally, adding at least 3 times of n-hexane, swirling, centrifuging, removing supernatant, and drying the precipitate to obtain the hyperbranched polymer with the end group modified by the group E, namely the cationic polyester.
In a third aspect the application discloses the use of the cationic polyesters of the application in nucleic acid drug delivery.
In one implementation of the application, nucleic acid agents include, but are not limited to, mRNA, loop RNA, siRNA, microRNA, saRNA, and DNA.
It should be noted that the key of the present application is that a novel nucleic acid drug delivery material, namely the cationic polyester of the present application, has been found; the cationic polyester disclosed by the application is used for delivering nucleic acid, so that the transfection efficiency is high, the cytotoxicity is low, and the clinical use requirement can be better met. It will be appreciated that the cationic polyesters of the present application, due to their low cytotoxicity, can be used not only for nucleic acid delivery, but also for other similar procedures requiring delivery of substances into cells.
In one implementation of the application, nucleic acid drug delivery comprises encapsulating a nucleic acid drug with the cationic polyester and delivering it into a cell.
In one implementation of the application, the cell types include, but are not limited to, HEK293T, A549, heLa, U87, HUVEC, jurkat, RAW264.7, iPSC, and MSC.
In a fourth aspect of the application, a nucleic acid delivery particle is disclosed, comprising a packaging material and a nucleic acid encapsulated within the packaging material; the wrapping material is the cationic polyester of the application or the wrapping material at least comprises the cationic polyester of the application; the nucleic acid is at least one of mRNA, loop RNA, siRNA, microRNA, saRNA and DNA.
The nucleic acid delivery particles of the application not only can improve transfection efficiency, but also have smaller cytotoxicity and better clinical application prospect due to the adoption of the cationic polyester of the application. It will be appreciated that the key to the nucleic acid delivery particles of the present application is the use of the cationic polyesters of the present application, and reference may be made to the prior art for specific entrapment methods and nucleic acid delivery methods.
It should be noted that the wrapping material of the present application may further include materials other than the cationic polyester of the present application according to different designs or requirements of the wrapping layer, and is not particularly limited herein. It will be appreciated that the transfection efficiency can be improved to some extent and cytotoxicity reduced as long as the cationic polyesters of the present application are contained.
In one implementation of the application, the nucleic acid delivery particles have a particle size of 30-500nm.
In a fifth aspect of the application, a kit for nucleic acid drug delivery is disclosed, comprising at least one of the following components:
(a) The cationic polyesters of the application;
(b) The nucleic acid delivery particles of the application.
The kit for delivering the nucleic acid drug can be a kit for embedding a specific nucleic acid drug, or can be the cationic polyester of the application, and a user designs and embeds the corresponding nucleic acid drug according to the requirement. It will be appreciated that the key to the kit of the application is the inclusion of the cationic polyester or nucleic acid delivery particles of the application, and that other conventional reagents required for nucleic acid delivery, such as some lipid or polymeric materials and the like, may be obtained with reference to the prior art or commercially available. Of course, for convenience of use, a part of the reagents may be combined into the kit of the present application, and are not particularly limited herein.
In a sixth aspect, the application discloses a method for improving transfection efficiency in nucleic acid drug delivery, comprising coating a nucleic acid drug with the cationic polyester of the application or a coating material comprising the cationic polyester of the application to form nucleic acid delivery particles, and cell-transfecting the nucleic acid drug with the nucleic acid delivery particles.
The method is characterized in that the cationic polyester is adopted to improve the transfection efficiency; in one implementation of the application, the cationic polyesters of the application have significantly better mRNA delivery efficiency than the original linear polymers and current optimal commercial mRNA transfection reagents, and lower cytotoxicity. In addition, the cationic polyester disclosed by the application is soluble in ethanol, can be better used for clinic, and has great practical application prospect. It will be appreciated that the method of the application for increasing transfection efficiency is critical to the use of the cationic polyesters of the application, and that reference is made to the prior art for other procedures and reagents for cell transfection and is not specifically limited herein.
The method for improving the transfection efficiency can directly adopt the cationic polyester as a packaging material, and can also add the cationic polyester into the existing packaging material; it will be appreciated that the transfection efficiency can be improved to varying degrees as long as the cationic polyesters of the present application are included.
The cationic polyesters of the application, or the nucleic acid delivery particles of the application, or the kits of the application can be used for the treatment of diseases for nucleic acid drug delivery; for example, when a nucleic acid drug is administered in vivo, the nucleic acid drug may be coated with the cationic polyester of the present application or a coating material containing the cationic polyester of the present application to form nucleic acid delivery particles, and the nucleic acid delivery particles are used for nucleic acid drug delivery; alternatively, the nucleic acid delivery particles of the present application are used directly to deliver nucleic acid drugs.
It should be noted that, the key point of the administration method of the present application is to use the cationic polyester or the nucleic acid delivery particles for nucleic acid drug delivery, and the specific operation of in vivo delivery and other auxiliary materials required can refer to the prior art, and are not specifically limited herein.
Due to the adoption of the technical scheme, the application has the beneficial effects that:
the cationic polyester of the application is prepared by adding the monomer T on the basis of three monomers of the monomer P, the monomer S and the monomer M or on the basis of two monomers of the monomer P and the monomer S, and polymerizing the monomer T with other monomers to obtain a novel polymer with a hyperbranched structure. The cationic polyester disclosed by the application is soluble in ethanol, and can be used for remarkably improving the transfection efficiency and meeting the clinical use requirements better due to lower cytotoxicity when being used for nucleic acid delivery.
Drawings
FIG. 1 is a synthetic route diagram of hyperbranched polymers and modification of end groups E in an embodiment of the application;
FIG. 2 is a graph of HBPA-E14-3 before and after end group modification in examples of the present application;
FIG. 3 is a GPC chart of HBPA-E14-1 and PACA-E14 in examples of the present application;
FIG. 4 is a quantitative plot of the end group modifications E14 of HBPA-E14-1 and PACA-E14 in an example of the application;
FIG. 5 is the results of particle size measurements of the complex of HBPA-E14-2 and mRNA in the examples of the present application;
FIG. 6 is a graph showing the results of mRNA transfection efficiency test of different HBPA-E in A549 and HEK 293T cells in the examples of the present application;
FIG. 7 shows the results of the test of HBPA-E14-3 modified by two different modification methods, CDI and DIC, and the transfection efficiency of mRNA under different mass ratio conditions in the examples of the present application;
FIG. 8 is a graph showing the results of DNA transfection efficiency test of different HBPA-E in HEK 293T cells in an example of the present application;
FIG. 9 is the results of an mRNA transfection efficiency test for HBPA-E modified with 26 different end groups E in A549 cells in the examples of the present application;
FIG. 10 is a graph showing the results of toxicity test of the polymer and mRNA complexes in the A549 cell model in the examples of the present application;
FIG. 11 shows the results of the test of the effect of mRNA transfection of HBPA-E in vivo in the examples of the present application.
Detailed Description
The existing commercial gene delivery materials have insufficient transfection efficiency and high toxicity, and greatly limit the wide application of gene therapy based on nucleic acid drug delivery. Although researches report that based on the in vitro synthesis biology technology, the biodegradable linear polymer obtained by catalyzing the polymerization of P, S, M three monomers by immobilized lipase has higher transfection efficiency and lower cytotoxicity; however, the transfection efficiency of the linear polymer is still not ideal, and the prepared gene delivery material is difficult to be used in practical clinic because it needs to be dissolved with a toxic organic solvent.
The application creatively adds a new monomer T on the basis of original three monomer polymerization, or replaces monomer M with monomer T, thereby preparing the novel cationic polyester with hyperbranched structure. Specifically, the cationic polyester is a hyperbranched polymer formed by polymerizing a monomer P, a monomer S and a monomer M or two monomers P and S with a monomer T, and the end group of the hyperbranched polymer is modified by a group E; monomer P is cyclic lactone; the monomer S is an organic acid with two or more carboxyl groups at the end group; the monomer M is a compound with two hydroxyl groups at the end group and one secondary amine or tertiary amine; the monomer T is a compound containing three or more hydroxyl groups; the end group modifying compound E which provides modification of the group E is a compound containing at least one primary, secondary or tertiary amine group.
The cationic polyester provided by the application has the advantages that the gene transfection efficiency is obviously improved, the cationic polyester is obviously superior to the original linear polymer and the existing optimal commercial mRNA transfection reagent, and the cytotoxicity is lower. More importantly, the cationic polyester disclosed by the application is soluble in ethanol, does not need to be dissolved by a toxic organic solvent, is easy to be amplified industrially, can better meet clinical use requirements, and has great practical application prospects.
The application is further illustrated by the following examples. The following examples are merely illustrative of the present application and should not be construed as limiting the application.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Examples
1. Synthesis of hyperbranched Polymer (HBPA) and modification of end group E (HBPA-E)
(1) HBPA synthesis
In the embodiment, a hyperbranched polymer (HBPA) formed by polymerizing P, S, M and T four monomers is adopted, and then the end group E of the HBPA is modified to obtain HBPA-E. Specifically, monomer P of this example adopts cyclopentadecanolide (PDL), monomer S adopts Sebacic Acid (SA), monomer M adopts Methyldiethanolamine (MDEA), monomer T adopts Triethanolamine (TEA), HBPA synthetic route is shown in (a) of FIG. 1, seven experiments and two controls are designed, raw materials are added into a round-bottom flask according to the feeding mole ratio of four monomers P (PDL)/S (SA)/M (MDEA)/T (TEA) in Table 1, immobilized lipase (CALB) is added according to 10wt% of the total raw material mass, and diphenyl ether of 200wt% of raw material is finally added. After 3 times replacement of the reaction system with argon, the reaction was continued for 48 hours at a pressure of 2.1mbar with stirring and heating to 90℃for 24 hours. After the completion of the reaction, the reaction mixture was filtered to remove lipase. Adding n-hexane into the filtrate, vortex and centrifugate, pouring out supernatant, adding dichloromethane into the precipitate to dissolve, adding n-hexane to separate out, centrifugate, and pouring out supernatant. The above operation was repeated 3 times. The precipitate was dried in vacuo for one day to give HBPA. Wherein the two control synthetic polymers are the linear polymers PACA.
TABLE 1 characterization of polymers synthesized with different feed ratios and their modified E14 products
In Table 1, "a" is calculated from 1H-NMR results, and "b" is calculated from GPC results.
(2) Modification of the terminal group E
This example uses end group modification compound E14 as shown in Table 1, and uses two different end group modification methods, namely N, N' -Carbonyldiimidazole (CDI) and Diisopropylcarbodiimide (DIC) as coupling agents, respectively, to carry out end group modification of the polymer.
The modification method comprises the following steps: using CDI as the coupling agent, the synthetic route was as shown in FIG. 1 (b), 250mg of HBPA was dissolved in 5mL of extra dry Dichloromethane (DCM) and 40eq of CDI was added with stirring. After the reaction system was replaced with argon 3 times, it was stirred at room temperature overnight. The reaction solution was concentrated to 3mL, 3 volumes of diethyl ether were added, and the mixture was vortexed and centrifuged to remove the precipitate. The supernatant was dried under reduced pressure, dissolved in extra dry DCM, and 40eq of E14 was added under stirring and reacted at room temperature for 24 hours. After the reaction, adding equal volume of deionized water into the reaction solution, and removing the upper water phase after vortex and centrifugal delamination. And adding the deionized water with the same volume, and repeating the operation for 5 times. To the lower dichloromethane solution was added 3 volumes of n-hexane, and the mixture was vortexed and centrifuged. The precipitate was dried in vacuo for one day to give HBPA-E14. PACA-E14 was obtained for control 1.
And a modification method II: using DIC as the coupling agent, the synthetic route was as shown in FIG. 1 (c), 250mg of HBPA was dissolved in 5mL of ultra-dry DCM, and 40eq of DIC and 40eq of E14 were added with stirring. After the reaction system was replaced with argon 3 times, it was stirred at room temperature overnight. After the reaction, adding equal volume of deionized water into the reaction solution, and removing the upper water phase by vortex and centrifugation. And adding the deionized water with the same volume, and repeating the operation for 3 times. Then adding 3 times of n-hexane into the dichloromethane at the lower layer, and swirling and centrifuging. The precipitate was washed with a small amount of ethanol and dried in vacuo for one additional day to give HBPA-E14, e.g., HBPA-E14-3-DIC as prepared in run 4. PACA-E14-DIC was obtained for control 2.
Using a method similar to that of test 3, this example further uses 25 end group modification compounds from E1 to E26 in addition to E14 to end group modify HBPA to synthesize other HBPA-E.
The 26 end group modifying compounds of E1 to E26 were as follows:
2. structural characterization of HBPA and HBPA-E
HBPA and HBPA-E can be characterized in terms of their molecular structure by 1H-NMR. The results are shown in Table 1, and the partial results are shown in FIG. 2. FIG. 2 shows the spectra of HBPA of test 2 before and after the end group modification, i.e., the 1H-NMR spectra of HBPA-3 and HBPA-E14-3, respectively, with CDCl as solvent 3 . In the spectrogram, the characteristic peak of the PDL unit is f (delta)About 4.08 ppm), the characteristic peak of SA unit is b (delta about 1.60 ppm), the characteristic peak of MDEA unit is g (delta about 4.20 ppm), and the characteristic peak of TEA unit is h (delta about 2.85 ppm). The presence of TEA units indicates that the polymer structure is in accordance with the expected assumption, that hyperbranched structures may be present, and that the characteristic peak k (delta. Apprxeq.2.22 ppm) may prove that the end groups have successfully modified the group E. The proportion of each repeating unit of the polymer can be obtained by calculating the integral area of each characteristic peak, and the result shows that the molar ratio of the P/S/M/T repeating units of the HBPA-E14-3 obtained by CDI modification is 1:9:4.5:1.9 and the feeding ratio of the P/S/M/T repeating units is 1:9:6:2 is very close. This means that the reaction of the monomer raw materials is substantially complete and the reaction efficiency is very high. The other 1H-NMR results are shown in Table 1. The results show that even with increasing feed T, the enzyme catalyzed reaction is nearly complete and the reaction can be very controlled to give hyperbranched polymers without cross-linking.
Experiments show that the reduction of the monomer M has less influence on the hyperbranched polymer; therefore, this example further investigated the case of reducing the ratio of monomer M to 0, i.e. directly using monomer P, monomer S and monomer T for the polymerization, in particular the feed molar ratio P: S: t=1:9:6, the remaining steps and parameters being identical to "(1) HBPA synthesis". The results show that the P, S and T monomers are also capable of obtaining polymers with hyperbranched structures without producing cross-links. P, S and T have similar physicochemical properties to the hyperbranched polymers obtained with P, S, M and T.
Meanwhile, the molecular weight and distribution of HBPA-E were characterized by GPC. GPC measurements were performed using a Waters 1515 column and 2414 Refractive Index (RI) detector at 35 ℃. The mobile phase was DMF containing 0.1% LiBr at a flow rate of 1mL/min, and a linear polymethyl methacrylate standard was used as standard. The results are shown in Table 1, and the partial results are shown in FIG. 3. FIG. 3 is a GPC chart of HBPA-E14-1 of test 3 and PACA-E14 of control 1, respectively. The results of FIG. 3 show that both the GPC patterns of hyperbranched HBPA-E14-1 and linear PACA-E14 are unimodal, number average molecular weight M n 8296 and 8769Da, respectively, and PDI of 4.12 and 1.97, respectively. The molecular weight of the two are close, but the PDI is differentObvious. This suggests that there is a significant structural difference between HBPA-E14-1 and PACA-E14, which should be due to the branched structure of HBPA. The PDI of the other HBPA-E14 is shown in Table 1 and is similar to that of HBPA-E14-1.
In addition, in this example, HBPA-E14 and PACA-E14 prepared in Table 1 were dissolved in ethanol and tested for solubility. The results are shown in Table 1. The results in Table 1 show that the branched HBPA-E of this example are all very soluble in ethanol, with a solubility of more than 25mg/mL, whereas the linear PACA-E is poorly soluble in ethanol, with a solubility of less than 10mg/mL.
Finally, the number average molecular weight similar HBPA-E14 (mn=8769) and PACA-E14 (mn=8296) end group modified E14 content were quantified by ninhydrin method in this example. The specific method comprises the following steps: 1. preparing an ninhydrin color former: 200mg of ninhydrin is weighed, dissolved in 9mL of ethanol, and ethanol is added to a volume of 10mL to prepare a 20mg/mL ninhydrin ethanol solution. 2. Sample measurement: about 10mg of HBPA-E14 and PACA-E14 are taken respectively to prepare 5mg/mL ethanol solutions, 100 mu L of each ethanol solution is mixed uniformly with 200 mu L of ninhydrin ethanol solution respectively, the mixture is placed on a shaking table, heated and oscillated at 60 ℃ for 10min, cooled to room temperature, 200 mu L of each ethanol solution is taken to measure the absorbance value at 570nm, and the absorbance value is converted into the relative concentration of end group E14. As a result, as shown in FIG. 4, the relative concentration of end group E14 in PACA-E14 was 1, while at the same material concentration, the relative concentration of end group E14 of HBPA-E14-1 was 3.01, indicating that the end group E14 content was approximately 3 times that of PACA-E14. Considering that the molecular weights of the two are very close, the significantly higher end group content of HBPA-E14-1 than PACA-E14 should be due to its branched structure, which again verifies the branched structure of HBPA.
3. Characterization of materials and mRNA complexes
After the preparation of the polymeric material HBPA-E14-2, it was mixed with Firefly Luciferase mRNA (N1-Me-pseudoUTP, nanjinouzan Biotechnology Co., ltd.) in different mass ratios and the diameter of its complex was determined, while also using the complex of PACA-E14 and mRNA as a control. Specifically, the polymer materials HBPA-E14-2 and PACA-E14 are respectively dissolved in ethanol and DMSO to prepare 25mg/mL polymer solutions, and then different amounts of the polymer solutions are added into a certain volume of NaAc buffer solution with pH of 4.9, and vortex shaking is performed. Simultaneously preparing NaAc solution with pH of 4.9 containing 20 mug/mL mRNA, adding an equal volume of polymer solution into the mRNA solution, and vortex mixing to finally obtain compound solutions with the mass ratio of the polymer to the mRNA of 25:1, 50:1, 75:1, 100:1, 150:1 and 200:1 respectively. The above mass ratios of HBPA-E14-2 and PACA-E14 were used as control tests, respectively. The amount of HBPA-E14-2 and PACA-E14 is calculated according to the mass ratio, for example, the mass ratio of the polymer to the mRNA is 25:1, then 20 mu L of 25mg/mL polymer solution, namely 500 mu g polymer is added into 980 mu L NaAc buffer solution with pH of 4.9, and vortex oscillation is carried out; simultaneously preparing NaAc solution with pH of 4.9 containing 20 mug/mL mRNA, adding an equal volume of polymer solution into the mRNA solution, and vortex mixing to obtain the mass ratio of the polymer to the mRNA of 25:1.
The composite particle size was tested by a particle sizer (Malvern Panalytical Zetasizer Pro) at 25 ℃. As a result, FIG. 5 shows that the abscissa of FIG. 5 shows the mass ratio of polymer to mRNA, and the ordinate shows the particle size of the complex in nm.
The results in FIG. 5 show that the HBPA-E14-2/mRNA complex is over 300nm in diameter at a mass ratio of 25:1, while the diameter is significantly reduced and gradually stabilizes at a mass ratio of 50:1-200:1, both 100-200nm. This suggests that the recombination of HBPA-E14-2 and mRNA tends to stabilize at mass ratios greater than 50:1. PACA-E14 and mRNA also showed similar results.
4. mRNA and DNA transfection efficiency test of HBPA-E in vitro cells
After obtaining a complex of HBPA-E and luciferase-expressing mRNA or DNA (Firefly Luciferase DNA, guangzhou Ai Ji Biotechnology Co., ltd.), the transfection efficiency was tested in different cell models. The specific method for cell transfection is as follows: inoculating cells (ATCC) such as A549 and HEK293T into a 48-well cell culture plate (2.5X10) 4 Well), cells at 37℃and containing 5% CO 2 After 12h of adherence in DMEM, the DMEM medium (250. Mu.L/well) was changed, and the samples to be tested and the positive control were added for 24h (mRNA) or 48h (DNA), respectively, and the samples were added as calculated from the final 2. Mu.g/mL mRNA per well. Preparation method of complex of material to be tested and mRNA or DNA is the same as "characterization of three, material and mRNA Complex" . The positive control for mRNA transfection was taken as Lipofectamine MessengerMAX (LipoMM) for Simeravid and Lipofectamine 2000 (Lipo 2 k) for DNA transfection.
The specific method for testing the lysis and luminous effect after cell transfection is as follows: cells were transfected for 24 hours (mRNA) or 48 hours (DNA) and medium in the well plate was aspirated, after which they were placed in a-80℃refrigerator for 10 minutes. Taking out the pore plate to place at 4 ℃, taking 40 mu L of lysate to a 48 pore plate, covering the bottom surface, standing for 5min, taking 280 mu L of test solution to the 48 pore plate, taking 160 mu L of mixture to a black enzyme-labeled instrument plate, and finally taking 40 mu L D-Luciferin by a gun and adding the mixture to each pore. After reacting for 2 minutes at normal temperature, placing the mixture into an enzyme-labeled instrument to test 560nm luminescence.
The results of the mRNA and DNA transfection efficiency tests of different HBPA-E in a549 and HEK293T cells are shown in fig. 6-9. The specific test designs are as follows:
in this example, the mRNA transfection efficiency in A549 and HEK293T cells, respectively, after the polymers of control 1, test 2, test 3, test 5, test 6 and test 7 were compounded with mRNA at a polymer to mRNA mass ratio of 75:1, respectively, was tested and the results are shown in FIG. 6.
The results of fig. 6 show that in a549 cells, all hyperbranched materials showed significantly higher mRNA transfection effect than the commercial control LipoMM, and far higher than that of linear PACA-E14. Wherein the transfection efficiency of HBPA-E14-3 is highest and is close to 4 times of LipoMM. Whereas linear PACA-E14 is less effective than LipoMM. The transfection results of HEK293T cells were consistent with the a549 results. This demonstrates that hyperbranched polymers have a significantly improved cell transfection effect on mRNA compared to linear polymers and are also superior to the current most efficient commercial transfection reagents, which effect is also demonstrated on different cells. Considering that this hyperbranched polymer is also soluble in ethanol, it has great potential for use in both in vitro and in vivo mRNA transfection.
Simultaneously, the HBPA-E14-3 modified by CDI and DIC and the mRNA transfection efficiency thereof under different mass ratio conditions are also tested in the example, specifically, the example respectively tests the transfection efficiency of the polymer and the mRNA of the CDI modified HBPA-E14-3 in A549 after the polymer and the mRNA of the test 3 are respectively compounded according to the mass ratio of 25:1, 50:1, 75:1 and 100:1, and the transfection efficiency of the polymer and the mRNA of the DIC modified HBPA-E14-3-DIC in A549 after the polymer and the mRNA of the test 4 are respectively compounded according to the mass ratio of 25:1, 50:1, 75:1 and 100:1; the results are shown in FIG. 7.
The results in FIG. 7 show that on A549 cells, HBPA-E14-3 modified in two ways had significantly higher transfection efficiency than LipoMM at mass ratios between 50:1 and 100:1, except that CDI modified material was better than DIC modified material. And at a mass ratio of 25:1, transfection efficiency was not exhibited. These results demonstrate that both modification methods are possible. While mRNA is not yet sufficiently complexed at 25:1, mRNA is already complexed above 50:1, consistent with the results of the particle size of the complex tested by the particle sizer.
In addition, the DNA transfection efficiency of different HBPA-E in HEK 293T cells was also tested in this example, specifically, the polymers of control 1, test 2, test 3, test 5, test 6 and test 7 were respectively tested and compounded with DNA according to the polymer to DNA mass ratio of 75:1, and the results are shown in FIG. 8.
The results of FIG. 8 show that all hyperbranched materials showed significantly higher DNA transfection effect than the commercial control Lipo2k and far higher than that of linear PACA-E14, with the transfection efficiency of HBPA-E14-2 being highest. This illustrates that hyperbranched polymeric materials can also be used for DNA transfection.
Finally, this example also tested the mRNA transfection efficiency in A549 cells of HBPA-E modified by modifying the end groups of 26 different end group modification compounds, specifically, tested the mRNA transfection efficiency in A549 cells after the end group modification of 26 kinds of HBPA-E by the end group modification compounds E1 to E26 are respectively compounded with mRNA according to the mass ratio of polymer to mRNA of 75:1, and the result is shown in FIG. 9.
The results of FIG. 9 show that different end group modifications have an effect on mRNA transfection efficiency, with a variety of end group modified HBPA-E exhibiting significantly higher transfection efficiencies than LipoMM, such as E2, E4, E9, E10, E12, E14, E15, E25, E26, and the like. Except that the transfection efficiency of the E11, E13, E22, E23 and E24 modified polymers on mRNA is obviously lower than that of lipoMM, the transfection efficiency of the E8, E17, E18 and E21 modified polymers on mRNA is equivalent to that of lipoMM, and the transfection efficiency of the rest end group modified polymers on mRNA is higher than that of lipoMM.
5. Cytotoxicity test
This example tested the cytotoxicity of its HBPA-E/mRNA complexes in the A549 cell model. The cytotoxicity test is specifically carried out as follows: a549 cells were seeded in 96-well cell culture plates (1×10) 4 After 12h adherence, the DMEM culture medium is replaced, and the mRNA complexes of the sample PACA-E14, HBPA-E14-3 and the positive control lipoMM to be detected are respectively added for 24h culture, wherein the mass ratio of the PACA-E14 or HBPA-E14-3 to the mRNA is 50:1. Samples were added at final mRNA concentrations of 0.125, 0.25, 0.5, 1, 2, 4 and 8. Mu.g/mL per well. After 24h, 10. Mu.L of CCK8 detection reagent was added to each well and the absorbance at 450nm was detected using a microplate reader. The results are shown in FIG. 10.
The results in fig. 10 show that the control LipoMM complex showed significant cytotoxicity, with only 50% cell viability at an mRNA concentration of 2 μg/mL, and with a significant increase in toxicity as the mRNA concentration was increased. In contrast, the toxicity of the linear PACA-E14 complex was significantly reduced, but also showed some cytotoxicity, which reached 70% cell viability at an mRNA concentration of 2. Mu.g/mL. Compared with the two, the toxicity of the hyperbranched HBPA-E14-3 complex is further reduced, and the cell survival rate is more than 80% at the mRNA concentration of 2 mug/mL. The result shows that the hyperbranched HBPA-E14-3 has higher biocompatibility and larger in vivo application prospect compared with commercial transfection reagents and linear PACA-E14.
6. Test of the effect of transfection of HBPA-E on mRNA in vivo
The effect of HBPA-E14-3 on mRNA transfection in vivo was tested in this example, and the specific method is as follows: c57 mice (weighing about 20 g) of 4-6 weeks old were purchased from medical laboratory animal center in Guangdong province, and monitored and raised at SPF (specific pathogen-free) environmental level. The HBPA-E14-3/mRNA complex was injected into mice by pulmonary administration (by insertion into the trachea with a pulmonary spray needle, 5. Mu.g dose) and intratracheal instillation administration (5. Mu.g dose), respectively, and the effect of luciferase expression in the mice was observed with physiological saline as a blank. The results are shown in FIG. 10. Wherein, in the HBPA-E14-3/mRNA complex, the mass ratio of the HBPA-E14-3 to the mRNA is 50:1.
In FIG. 11, the abscissa "Control" represents a blank, "pulmonary administration of mRNA (5. Mu.g)" represents pulmonary administration directly with mRNA, "pulmonary administration (5. Mu.g)" represents pulmonary administration with HBPA-E14-3/mRNA complex, "intratracheal instillation (5. Mu.g)" represents intratracheal instillation with HBPA-E14-3/mRNA complex.
The results of fig. 11 show that neither direct mRNA pulmonary administration nor the blank detected fluorescence, whereas the HBPA-E14-3/mRNA complex shows a significant luciferase expression effect from different routes of administration, where pulmonary administration is 2 times the delivery efficiency of intratracheal instillation. These results all indicate that the hyperbranched polymer/mRNA compound can realize mRNA delivery in vivo and has great application prospect in vivo.
The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the application.
Claims (37)
1. A cationic polyester characterized in that: the cationic polyester is a hyperbranched polymer formed by polymerizing a monomer P, a monomer S and a monomer M or two monomers P and S with a monomer T, and the end group of the hyperbranched polymer is modified by a group E;
wherein, monomer P is cyclic lactone;
the monomer S is an organic acid with two or more carboxyl groups at the end group;
the monomer M is a compound containing two hydroxyl groups and one secondary amine or tertiary amine;
the monomer T is a compound containing three or more hydroxyl groups;
the end group modifying compound E which provides modification of the group E is a compound containing at least one primary, secondary or tertiary amine group.
2. The cationic polyester according to claim 1, characterized in that: monomer P is a cyclic lactone having a fatty chain length of 6 to 35.
3. The cationic polyester according to claim 2, characterized in that: the monomer P is at least one of cyclohexanolactone, cyclododecanolactone, cyclopentadecanolide and cyclohexadecanolide.
4. The cationic polyester according to claim 1, characterized in that: monomer S is an organic acid with a carbon chain length of 3 to 18.
5. The cationic polyester according to claim 4, wherein: the monomer S is at least one of adipic acid, sebacic acid and 1,2, 3-propane tricarboxylic acid.
6. The cationic polyester according to claim 1, characterized in that: monomer M is a compound having a carbon chain length of 4 to 36.
7. The cationic polyester according to claim 6, wherein: the monomer M is at least one of diethanolamine, methyl diethanolamine and ethyl diethanolamine.
8. The cationic polyester according to claim 1, characterized in that: monomer T is a compound having a carbon chain length of 4 to 54.
9. The cationic polyester of claim 8, wherein: the monomer T is at least one of trimethylolpropane, 3- (hydroxymethyl) -1, 5-pentanediol, triethanolamine, N, N, N ', N' -tetrahydroxyethyl ethylenediamine.
10. The cationic polyester according to claim 1, characterized in that: providing a group E modified end group modifying compound E as at least one of E1 to E26;
。
11. the cationic polyester according to claim 10, characterized in that: the end group modification compound E provided with the group modification is at least one of E1 to E10, E12, E14 to E21, E25 and E26.
12. The cationic polyester of claim 11, wherein: the end group modification compound E provided with the group modification is at least one of E2, E4, E9, E10, E12, E14, E15, E25 and E26.
13. The cationic polyester according to any one of claims 1 to 12, characterized in that: the cationic polyester is a hyperbranched polymer with a structure shown in a formula I;
one (I)
Wherein x, y, z are independent integers from 1 to 200,
n is an integer of 0 to 200,
j. k is an integer of 0 to 30,
l, m, o, p, q is an independent integer from 1 to 20,
R x is hydrogen, or a substituted or unsubstituted alkyl group having 1 to 18 carbon atoms, or a substituted or unsubstituted aryl group having at least 1 benzene ring, or a substituted or unsubstituted heterocyclic group having at least 1 heterocyclic ring, or a substituted or unsubstituted alkoxy group having 1 to 18 carbon atoms and at least 1 oxygen atom;
j is hydrogen, R 1 Modification of the group E provided for the end group-modifying compound E, in which case there is no R 2 The method comprises the steps of carrying out a first treatment on the surface of the Alternatively, J is carbonyl, in which case R 1 And R is 2 Both are the group E modifications provided by the end group modifying compound E;
in the formula I, the broken line represents a branch structure, the first monomer connected with the branch structure is P or S, and other monomers are connected subsequently to form a hyperbranched structure.
14. The cationic polyester according to any one of claims 1 to 12, characterized in that: the cationic polyesters have a number average molecular weight of from 1 to 30 k.
15. The cationic polyester of claim 14, wherein: the cationic polyesters have a number average molecular weight of from 2 to 20 k.
16. The process for preparing a cationic polyester according to any one of claims 1 to 15, characterized in that: adding monomers and a catalyst into a solvent, sequentially carrying out a first-stage polymerization reaction and a second-stage polymerization reaction in an inert atmosphere, and removing the catalyst after the reaction is finished to obtain a hyperbranched polymer; then, under the action of a coupling agent, adopting an end group modification compound E to carry out end group modification on the hyperbranched polymer to obtain cationic polyester with end groups modified by the group E;
the conditions of the first-stage polymerization reaction are that the temperature is 85-95 ℃, the reaction vacuum degree is 50-1000 mbar, and the reaction time is 12-24 hours;
the conditions of the second-stage polymerization reaction are that the temperature is 85-95 ℃, the reaction vacuum degree is 1-30 mbar, and the reaction time is 12-72 h.
17. The method of manufacturing according to claim 16, wherein: the molar ratio of the monomer P to the monomer S is 0.1:10-4:1, the molar ratio of the monomer M to the monomer S is 0:10-20:10, and the molar ratio of the monomer T to the monomer S is 0.1:10-20:10.
18. The method of manufacturing according to claim 16, wherein: the catalyst is immobilized lipase.
19. The method of manufacturing according to claim 18, wherein: the dosage of the immobilized lipase is 3-50 wt% of the total mass of each monomer.
20. The method of manufacturing according to claim 16, wherein: the solvent is at least one of diphenyl ether, n-dodecane, 1-butyl-3-methylimidazole hexafluorophosphate, dimethylacetamide and phthalic dimethyl ether.
21. The method of manufacturing according to claim 20, wherein: the solvent is used in an amount of 100-500 wt% of the total mass of each monomer.
22. The method of manufacturing according to claim 16, wherein: the catalyst removal specifically comprises the steps of filtering reaction liquid by a filtering device after the reaction is finished, and collecting filtrate; adding n-hexane into the filtrate to separate out the hyperbranched polymer, centrifuging, removing supernatant, adding dichloromethane into the precipitate to dissolve, adding n-hexane to separate out the hyperbranched polymer, centrifuging, removing supernatant, and repeatedly dissolving with dichloromethane, separating out n-hexane and centrifuging for at least 2 times; finally, the precipitate is dried, and the hyperbranched polymer is obtained.
23. The method of manufacturing according to claim 16, wherein: the coupling agent is at least one of N, N' -carbonyl diimidazole, carbodiimide, phosphorus positive ions and urea positive ions.
24. The method of manufacturing according to claim 23, wherein: the carbodiimide class includes diisopropylcarbodiimide, dicyclohexylcarbodiimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide;
the phosphorus positive ion class comprises benzotriazol-1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate;
the urea positive ion class includes 2- (7-azabenzotriazol) -N, N '-tetramethylurea hexafluorophosphate and O-benzotriazol-N, N' -tetramethylurea tetrafluoroboric acid.
25. The method of manufacturing according to claim 23, wherein: the molar ratio of the coupling agent to the hyperbranched polymer is 2:1-50:1.
26. The method of manufacturing according to claim 25, wherein: the molar ratio of the coupling agent to the hyperbranched polymer is 4:1-15:1.
27. The method of manufacturing according to claim 23, wherein: the molar ratio of the coupling agent to the end group modification compound E is 5:1-1:10.
28. The method of any one of claims 16-27, wherein: the end group modification compound E is adopted to carry out end group modification on the hyperbranched polymer, and concretely comprises the steps of dissolving the hyperbranched polymer in dichloromethane, adding a coupling agent, stirring at least 10 h at room temperature in an inert atmosphere, concentrating a reaction solution, adding diethyl ether with the volume of at least 3 times, centrifuging, removing sediment, and obtaining supernatant; removing the solvent in the supernatant, adding the dried product into dichloromethane to dissolve, adding an end group modification compound E under stirring, and reacting at room temperature for at least 10 h to obtain the hyperbranched polymer with the end group modified by the group E, namely the cationic polyester.
29. The method of manufacturing according to claim 28, wherein: after the end group modification compound E is added and the room temperature reaction is finished, adding equal volume of deionized water into the reaction liquid, and removing an upper water phase after vortex and centrifugal layering; repeatedly adding equal volume of deionized water, swirling, centrifuging for layering, and removing upper water phase for at least 3 times; and finally, adding at least 3 times of n-hexane, swirling, centrifuging, removing supernatant, and drying the precipitate to obtain the hyperbranched polymer with the end group modified by the group E.
30. Use of a cationic polyester according to any one of claims 1-15 in nucleic acid drug delivery.
31. The use according to claim 30, wherein: the nucleic acid drugs include mRNA, loop RNA, siRNA, microRNA, saRNA, and DNA.
32. The use according to claim 30 or 31, characterized in that: the nucleic acid drug delivery includes encapsulating a nucleic acid drug with the cationic polyester and delivering it into a cell.
33. The use according to claim 32, characterized in that: the cell types include HEK293T, A549, heLa, U87, HUVEC, jurkat, RAW 264.7.264.7, iPSC and MSC.
34. A nucleic acid delivery particle, characterized in that: comprises a wrapping material and nucleic acid wrapped in the wrapping material;
the wrapper comprising the cationic polyester of any one of claims 1-15;
the nucleic acid is at least one of mRNA, loop RNA, siRNA, microRNA, saRNA and DNA.
35. The nucleic acid delivery particle of claim 34, wherein: the nucleic acid delivery particles have a particle size of 30-500 nm.
36. A kit for nucleic acid drug delivery, characterized in that: comprising at least one of the following components,
(a) The cationic polyester of any one of claims 1-15;
(b) The nucleic acid delivery particle of claim 34 or 35.
37. A method for increasing transfection efficiency in nucleic acid drug delivery, characterized by: comprising encapsulating a nucleic acid drug with a cationic polyester according to any one of claims 1 to 15 or an encapsulating material comprising a cationic polyester according to any one of claims 1 to 15 to form a nucleic acid delivery particle, and cell-transfecting the nucleic acid drug with the nucleic acid delivery particle.
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