CN116236584B - Polysaccharide-polypeptide conjugate for high-efficiency siRNA delivery - Google Patents
Polysaccharide-polypeptide conjugate for high-efficiency siRNA delivery Download PDFInfo
- Publication number
- CN116236584B CN116236584B CN202310133398.5A CN202310133398A CN116236584B CN 116236584 B CN116236584 B CN 116236584B CN 202310133398 A CN202310133398 A CN 202310133398A CN 116236584 B CN116236584 B CN 116236584B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- lip
- sirna
- polypeptide
- polypeptide conjugate
- 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.)
- Active
Links
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 50
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 41
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims abstract description 55
- 229920001287 Chondroitin sulfate Polymers 0.000 claims abstract description 53
- 229940059329 chondroitin sulfate Drugs 0.000 claims abstract description 53
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 18
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 17
- 125000002091 cationic group Chemical group 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 239000002502 liposome Substances 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- 230000004913 activation Effects 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 12
- 238000007906 compression Methods 0.000 claims description 10
- 230000006835 compression Effects 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 150000001408 amides Chemical class 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 5
- 229920002873 Polyethylenimine Polymers 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 229920001661 Chitosan Polymers 0.000 claims description 2
- 108010039918 Polylysine Proteins 0.000 claims description 2
- 102000007327 Protamines Human genes 0.000 claims description 2
- 108010007568 Protamines Proteins 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000000412 dendrimer Substances 0.000 claims description 2
- 229920000736 dendritic polymer Polymers 0.000 claims description 2
- 229920000656 polylysine Polymers 0.000 claims description 2
- 229920000193 polymethacrylate Polymers 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- 229940048914 protamine Drugs 0.000 claims description 2
- 229920001601 polyetherimide Polymers 0.000 claims 1
- 238000001727 in vivo Methods 0.000 abstract description 12
- 210000003660 reticulum Anatomy 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 6
- PAPYICJQRHSQGK-UHFFFAOYSA-N 4-(2,5-dichloro-thiophen-3-yl)-pyrimidin-2-ylamine Chemical compound NC1=NC=CC(C2=C(SC(Cl)=C2)Cl)=N1 PAPYICJQRHSQGK-UHFFFAOYSA-N 0.000 description 58
- 210000004027 cell Anatomy 0.000 description 38
- 230000008685 targeting Effects 0.000 description 22
- 230000000694 effects Effects 0.000 description 19
- 210000004185 liver Anatomy 0.000 description 19
- 210000004024 hepatic stellate cell Anatomy 0.000 description 15
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 13
- 239000002105 nanoparticle Substances 0.000 description 12
- 238000001994 activation Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 230000004048 modification Effects 0.000 description 11
- 238000012986 modification Methods 0.000 description 11
- 230000014509 gene expression Effects 0.000 description 10
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 10
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 9
- 208000019425 cirrhosis of liver Diseases 0.000 description 9
- 230000030279 gene silencing Effects 0.000 description 9
- 238000012226 gene silencing method Methods 0.000 description 9
- 239000002114 nanocomposite Substances 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Natural products OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 8
- 230000004900 autophagic degradation Effects 0.000 description 8
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 8
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 230000037361 pathway Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 102100032912 CD44 antigen Human genes 0.000 description 6
- 206010016654 Fibrosis Diseases 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 6
- 230000002300 anti-fibrosis Effects 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 230000004761 fibrosis Effects 0.000 description 6
- 230000009368 gene silencing by RNA Effects 0.000 description 6
- 210000002288 golgi apparatus Anatomy 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 230000032258 transport Effects 0.000 description 6
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 5
- 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 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 102000016613 Autophagy-Related Protein 7 Human genes 0.000 description 4
- 108010092778 Autophagy-Related Protein 7 Proteins 0.000 description 4
- 102000012422 Collagen Type I Human genes 0.000 description 4
- 108010022452 Collagen Type I Proteins 0.000 description 4
- 235000021314 Palmitic acid Nutrition 0.000 description 4
- 210000001130 astrocyte Anatomy 0.000 description 4
- 230000004700 cellular uptake Effects 0.000 description 4
- 239000010408 film Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 230000010189 intracellular transport Effects 0.000 description 4
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 4
- 229960003471 retinol Drugs 0.000 description 4
- 235000020944 retinol Nutrition 0.000 description 4
- 239000011607 retinol Substances 0.000 description 4
- YZOUYRAONFXZSI-SBHWVFSVSA-N (1S,3R,5R,6R,8R,10R,11R,13R,15R,16R,18R,20R,21R,23R,25R,26R,28R,30R,31S,33R,35R,36R,37S,38R,39S,40R,41S,42R,43S,44R,45S,46R,47S,48R,49S)-5,10,15,20,25,30,35-heptakis(hydroxymethyl)-37,39,40,41,42,43,44,45,46,47,48,49-dodecamethoxy-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.23,6.28,11.213,16.218,21.223,26.228,31]nonatetracontane-36,38-diol Chemical compound O([C@@H]([C@H]([C@@H]1OC)OC)O[C@H]2[C@@H](O)[C@@H]([C@@H](O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3O)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O3)O[C@@H]2CO)OC)[C@H](CO)[C@H]1O[C@@H]1[C@@H](OC)[C@H](OC)[C@H]3[C@@H](CO)O1 YZOUYRAONFXZSI-SBHWVFSVSA-N 0.000 description 3
- -1 CK1 compound Chemical class 0.000 description 3
- 229920000858 Cyclodextrin Polymers 0.000 description 3
- 206010019668 Hepatic fibrosis Diseases 0.000 description 3
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- XSDQTOBWRPYKKA-UHFFFAOYSA-N amiloride Chemical compound NC(=N)NC(=O)C1=NC(Cl)=C(N)N=C1N XSDQTOBWRPYKKA-UHFFFAOYSA-N 0.000 description 3
- 229960002576 amiloride Drugs 0.000 description 3
- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 description 3
- 229960001076 chlorpromazine Drugs 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 230000003176 fibrotic effect Effects 0.000 description 3
- 230000037406 food intake Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000001476 gene delivery Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 230000006655 lysosomal degradation pathway Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000003463 organelle Anatomy 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 229920002567 Chondroitin Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108700023372 Glycosyltransferases Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 206010067125 Liver injury Diseases 0.000 description 2
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 210000001056 activated astrocyte Anatomy 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000020411 cell activation Effects 0.000 description 2
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 2
- KXKPYJOVDUMHGS-OSRGNVMNSA-N chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 2
- 230000006395 clathrin-mediated endocytosis Effects 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000002121 endocytic effect Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000012637 gene transfection Methods 0.000 description 2
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 description 2
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 231100000753 hepatic injury Toxicity 0.000 description 2
- 230000000887 hydrating effect Effects 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 210000004500 stellate cell Anatomy 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- KDLSKKYZXNKGTK-LQDDAWAPSA-M trimethyl-[(z)-2-[(z)-octadec-9-enoyl]-4-oxohenicos-12-enyl]azanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)CC(C[N+](C)(C)C)C(=O)CCCCCCC\C=C/CCCCCCCC KDLSKKYZXNKGTK-LQDDAWAPSA-M 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 229940045997 vitamin a Drugs 0.000 description 2
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 102000016726 Coat Protein Complex I Human genes 0.000 description 1
- 108010092897 Coat Protein Complex I Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000002337 anti-port Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 230000027448 caveolin-mediated endocytosis Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 101150076615 ck gene Proteins 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000008290 endocytic mechanism Effects 0.000 description 1
- 230000004651 endocytosis pathway Effects 0.000 description 1
- 230000030583 endoplasmic reticulum localization Effects 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000037440 gene silencing effect Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003547 hepatic macrophage Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007762 localization of cell Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000003986 lysosome degradation Effects 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 230000006491 negative regulation of transport Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 230000030589 organelle localization Effects 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 229920000962 poly(amidoamine) Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000001743 silencing effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 230000003746 surface roughness Effects 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000007723 transport mechanism Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000028973 vesicle-mediated transport Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a polysaccharide-polypeptide conjugate for efficiently delivering siRNA, which is KDEL polypeptide grafted chondroitin sulfate (CK), and has the structural formula:wherein a and b are positive integers more than or equal to 1, R is SO 3 ‑ . The polysaccharide-polypeptide conjugate is a component of a carrier for efficiently delivering siRNA in vitro and in vivo, and realizes efficient in-vivo and in-vitro gene interference treatment through a CD 44-Golgi-endoplasmic reticulum transport path.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a polysaccharide-polypeptide conjugate for efficiently delivering siRNA.
Background
RNA interference (RNAi) refers to the process by which double-stranded RNA molecules (dsRNA) specifically degrade mRNA and prevent its translation into protein. The expression of any gene can be reversibly silenced by simply changing the sequence of the siRNA, which is of great importance for some "drug-free treatable" diseases. The first siRNA drug ONPATTRO was approved by FDA in 2018 TM Research into vectors for efficient siRNA delivery is receiving increasing attention. Cytoplasmic delivery of siRNA drugs is critical for their effective gene silencing. In particular, the endosomal-lysosomal pathway is the homing of most gene delivery vectors after entry into the cell, endosomal escape being a prerequisite for their cytoplasmic delivery. Studies have shown that using lipid nanoparticles to deliver siRNA, only 1-2% of the siRNA can escape from the endosome into the cytoplasm, which hampers the gene transfection effect. Based on this, we have a need for an siRNA delivery vector that can circumvent the endosome-lysosomal degradation pathway, enabling efficient gene interference. Inspired by the mechanism of some viruses transfected cells, the change of the intracellular transport path of nanoparticles provides a new idea for designing a novel and effective gene delivery system.Jiamang Wang et al designed endoplasmic reticulum membrane-coated hybrid nanoparticles that could alter the intracellular transport pathway of siRNA through the endosomal-golgi-endoplasmic reticulum pathway, enhancing siRNA delivery efficiency. Meanwhile, since the endoplasmic reticulum membrane is connected with the nuclear membrane (acting site of DNA), ribosomes (acting site of mRNA and siRNA) are attached, and the endoplasmic reticulum targeted gene delivery system has the advantage of space. The above studies suggest that we turn the intracellular delivery route of gene drugs, designing a delivery vehicle with a golgi/endoplasmic reticulum transport route, would make it possible to achieve efficient siRNA delivery. CD44 is highly expressed on activated astrocytes, various tumor cells (e.g., breast cancer, melanoma, colon cancer, liver cancer, acute myeloid lymphomas, etc.), and thus CD 44-mediated targeting is widely used in the design of active drug delivery systems. Studies have shown that chondroitin sulfate is able to target the CD44 receptor, targeting the Golgi apparatus after uptake by cells, while bypassing the lysosomal degradation pathway. To further achieve endoplasmic reticulum targeting of gene vectors, we designed KDEL grafted chondroitin sulfate CK. Modification of the KDEL peptide segment facilitates transport of the gene vector from the Golgi apparatus to the endoplasmic reticulum via the COP I envelope vesicle. In summary, we assume that KDEL grafted chondroitin sulfate will be able to bypass the classical lysosomal degradation pathway by the CD 44-golgi-endoplasmic reticulum transport pathway, enabling efficient gene transfection.
Activation of hepatic stellate cells is a core event of liver fibrosis. During liver fibrosis, hepatic stellate cells are energized by autophagy to meet the energy required for their activation, so specific inhibition of autophagy to activate hepatic stellate cells would bring new promise for the treatment of liver fibrosis. However, the lack of autophagy-specific drugs and the high reliance on cell population targeting limit the use of autophagy-targeted anti-hepatic fibrosis therapies. siRNA mediated RNA interference provides hope for designing target drugs with high efficiency and specificity.
In the invention, we use autophagy interference siRNA (siATG 7) as a model siRNA, activated hepatic stellate cells as a cell model, hepatic fibrosis as an in-vivo treatment model, and verify and evaluate the in-vitro gene interference efficiency and treatment effect of the polysaccharide-polypeptide conjugate CK modified gene vector Lip/siATG 7/CK.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a polysaccharide-polypeptide conjugate CK capable of being used for delivering siRNA for realizing effective gene interference treatment, and the conjugate CK can realize cascade targeting of CD 44-Golgi body-endoplasmic reticulum and avoid a lysosome degradation path.
The aim of the invention is realized by the following technical scheme:
the polysaccharide-polypeptide conjugate is KDEL polypeptide grafted chondroitin sulfate, and has the structural formula:
wherein a and b are positive integers more than or equal to 1, R is SO 3 - 。
The preparation method of the polysaccharide-polypeptide conjugate comprises the following steps:
chondroitin sulfate, EDC and NHS are dissolved in PBS buffer solution for activation, then KDEL polypeptide is added, the pH is regulated to 7-8, and the KDEL polypeptide is grafted on the chondroitin sulfate through amide reaction.
The EDC and NHS have the function of improving the coupling efficiency, and can be replaced by a reaction system such as DCC/DIC/BOP/HOBT/CDI/HATU/HBTU.
The activation temperature is 10-45 ℃; and/or the temperature of the amide reaction is 10-45 ℃, and the time of the amide reaction is 0.5-72h. Preferably, the amide reaction time is 48 hours.
The structural formula of the chondroitin sulfate is as follows:R＝SO 3 - sodium salts, namely sodium chondroitin sulfate (CAS number: 39455-18-0), are generally used.
The structural formula of the KDEL polypeptide is as follows:
further, the molar ratio of chondroitin sulfate to KDEL polypeptide is 1:0.2-1; preferably, the molar ratio is 1:0.5-0.75.
Further, the molar ratio of chondroitin sulfate, EDC and NHS is 1:1 to 5:0.5 to 5; preferably, the molar ratio is 1:2.5:1.5.
further, the activation temperature is 10-45 ℃; preferably, the activation temperature is 37 ℃;
and/or the temperature of the amide reaction is 10-45 ℃ and the reaction time is 0.5-75h; preferably, the reaction temperature is 37 ℃.
The polysaccharide-polypeptide conjugates are useful for the entrapment and delivery of siRNA in the cytoplasm.
The siRNA includes, but is not limited to siATG7.
Further, when the polysaccharide-polypeptide conjugate is used to deliver siATG7, the KDEL has a grafting ratio of 5-90%; preferably, the grafting rate of KDEL is 10-15%.
Further, the method for loading siRNA on the carrier comprises the following steps:
1) Preparing a cationic gene Compression Vector (CV);
2) Mixing siRNA with a cationic gene compression carrier to form CV/siRNA complex;
3) The polypeptide-polysaccharide conjugate is dissolved in PBS, and then CV/siRNA complex is dripped into the solution to form CV/siRNA/CK.
Further, in step 2), the molar ratio of N of the cationic gene compression vector to P in the siRNA is greater than 4:1.
Further, in step 3), the addition amount of the polysaccharide-polypeptide conjugate CK is as follows: the molar ratio of-COOH on chondroitin sulfate backbone in CK to N in CV/siRNA complex is 0.6-3.6:1, a step of; preferably, the molar ratio is 2.8-3.6:1. optimally, the molar ratio is 2.8:1.
further, in step 1), the cationic gene compression carrier comprises one of cationic liposome/Lip, polyethyleneimine/PEI and derivatives thereof, polymethacrylate, DGL, polyamide-amine dendrimer/PAMAM, polylysine/PLL, chitosan and derivatives thereof, or protamine.
Preferably, the cationic gene compression vector is a cationic liposome.
Further, the specific method for preparing the cationic liposome comprises the following steps: DOTAP (2, 3-dioleoyl-propyl) -trimethylammonium-chloride; (2, 3-dioleoyl-propyl) -trimethyl ammonium chloride and CHO (cholesterol) in a molar ratio of 0.3-1:1 in chloroform, then volatilizing chloroform by a rotary evaporator to form a transparent film, adding DEPC water, hydrating for 30 minutes at 55 ℃, and homogenizing the liposome using probe ultrasound.
The beneficial effects of the invention are as follows:
the invention constructs the polysaccharide-polypeptide conjugate CK with the CD 44-Golgi-endoplasmic reticulum transport characteristic for in vivo RNA interference treatment. CK modified siRNA delivery systems will fulfill the following functions: 1. hepatic stellate cells (and various cells expressed by CD44, such as melanoma cells, breast cancer cells, etc.) are targeted and activated by CD44 mediated targeting. 2. Targeting the golgi via the cryptamine-mediated endocytic pathway while bypassing the lysosomal degradation pathway. 3. Endoplasmic reticulum localization is achieved by COPI vesicle-mediated antiport. Finally, the CK modified nano-composite (Lip/siATG 7/CK) conforms to an intracellular transport mechanism, and targets an endoplasmic reticulum downstream, so that a high-efficiency in-vivo RNAi treatment effect is realized.
Compared with the chondroitin sulfate modified nano-composite Lip/siATG7/CS, lip/siATG7/CK shows better in-vitro transfection effect and anti-fibrosis effect. In addition, CD44 overexpression is not limited to activating hepatic stellate cells, but most tumor cells also overexpress CD44 receptor, so CK has potential for siRNA treatment of various diseases associated with CD44 overexpression.
Drawings
FIG. 1 is a diagram of the construction and intracellular delivery pathways of KDEL grafted chondroitin sulfate;
FIG. 2 is a graph showing the results of examining particle diameters (A), potentials (B) and cell uptake (C) of Lip/siATG7/CS and Lip/siATG7/CK1 in experimental example 1 at different C/N ratios;
FIG. 3 is a graph showing the gene silencing efficacy test (D) uptake (E) and uptake mechanism test (F) of different C/N ratios Lip/siATG7/CK1 in experimental examples 2 and 3;
FIG. 4 shows the gene silencing efficacy and half-quantification of WB (C-E) of each of the gene nanocomposites by q-PCR (A) and WB (B) at a C/N ratio of 2.8:1 in experimental example 4. Effects of siNC-loaded formulations on protein expression (F);
FIG. 5 shows the results of immunofluorescence (A), scratch assay, semi-quantitative (B), and fat droplet metabolism assay (C) of Experimental examples 4-5;
FIG. 6 shows the result (A) of the co-localization experiment of organelles in Experimental example 6 and the result (B) of the intracellular release investigation in Experimental example 7;
FIG. 7 is a graph (A) showing intracellular transport mechanism, a study (B) of transport path, and an evaluation result (C) of the effect of inhibition of transport path on gene silencing efficiency in Experimental example 8;
FIG. 8 is an in vivo targeting and astrocyte targeting study in Experimental example 9;
FIG. 9 shows the dosing regimen examined for in vivo anti-fibrosis in Experimental example 10 and the experimental results.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings, but the scope of the present invention is not limited to the following description.
Example 1
The preparation method of the chondroitin sulfate-KDEL conjugate comprises the following specific steps:
dissolving Chondroitin Sulfate (CS), EDC and NHS in PBS according to the mol ratio of 1:2.5:1.5, activating for 20min at 37 ℃, adding KDEL polypeptide according to the mol ratio of CS to KDEL of 2:1, adjusting pH to 7-8, reacting for 48h at 37 ℃, removing impurities by a dialysis method, and freeze-drying to obtain CK1, wherein the grafting rate of the polypeptide is 11% (calculated as CK 1) calculated by nuclear magnetic resonance hydrogen spectrum.
Example 2
Referring to example 1, a chondroitin sulfate-KDEL conjugate was prepared as follows:
dissolving chondroitin sulfate, EDC and NHS in PBS according to the mol ratio of 1:2.5:1.5, activating for 20min at 37 ℃, adding KDEL polypeptide according to the mol ratio of CS to KDEL of 4:3, adjusting pH to 7-8, reacting for 48h at 37 ℃, removing impurities by a dialysis method, and freeze-drying to obtain CK2, wherein the grafting rate of the polypeptide is 40% (calculated as CK 2) by nuclear magnetic resonance hydrogen spectrum.
Example 3
The preparation method of the gene nano-composite comprises the following steps: cationic liposomes (Lip) were prepared by thin film dispersion. The liposome has a particle size of 108.9+ -0.6, a potential of 56.3+ -0.2, and PDI of 0.231+ -0.001. When DOTAP and siRNA (n/n) are larger than 75:1, the siRNA can be completely compressed by the liposome, the molar ratio of 150:1 is selected, and the cationic liposome is used for compressing the siRNA to form Lip/siATG7. And respectively dripping the Lip/siATG7 compound into CS, CK1 and CK2 solutions under vortex to respectively prepare Lip/siATG7/CS, lip/siATG7/CK1 and Lip/siATG7/CK2 gene nano-composites.
Experimental example 1
Examine different C/N ratio modified Lip/siATG7/CS and Lip/siATG7/CK1 particle size, potential and cellular uptake:
cationic liposomes (Lip) were prepared by thin film dispersion. The liposome has a particle size of 108.9+ -0.6, a potential of 56.3+ -0.2, and PDI of 0.231+ -0.001. When DOTAP and siRNA (n/n) are larger than 75:1, the siRNA can be completely compressed by the liposome, the molar ratio of 150:1 is selected, and the Lip/siATG7 compound is dripped into the CK1 solution under vortex to prepare the Lip/siATG7/CK1 compound.
The C/N ratio is defined as the molar ratio of carboxyl groups on the chondroitin sulfate backbone to DOTAP. At a C/N ratio of less than 0.6:1, insoluble polymers are formed. Gene nanocomposites were prepared according to example 3. When the ratio of C/N is 0.6:1-3.6:1, the Lip/siATG7/CK1 forms a stable compound, and the particle size is about 220 nm. As the C/N ratio increases, the potential increases from-20 mV to-40 mV. To screen for the optimal C/N ratio, HSC-T6 cell uptake of different C/N ratio complexes was determined.
The film dispersing method comprises the following specific steps: DOTAP and CHO were mixed in a molar ratio of 1:1 in chloroform, evaporating chloroform at 37deg.C under rotation at 100rpm to form a uniform transparent film, adding DEPC water, hydrating lipid film at 55deg.C under 180rpm with shaking table, and homogenizing the liposome (80W, 3 min) with probe ultrasound under ice bath.
The specific preparation methods of the Lip/siATG7/CK1, lip/siATG7/CK2 and Lip/siATG7/CS compound are as follows: mixing siATG7 and liposome in 75uL system DEPC water according to a molar ratio of 150:1 and incubating for 15min to form Lip/siRNA complex; and (3) respectively dissolving CK1, CK2 and CS in 25uLPBS, respectively dripping the Lip/siRNA complex into CK1, CK2 and CS solutions under vortex conditions, and incubating at room temperature for 15min to respectively form Lip/siRNA/CK1, lip/siATG7/CK2 and Lip/siATG7/CS.
The method for measuring the relative uptake rate of cells comprises the following steps: lip/FAM-siNC/CK1, lip/FAM-siNC/CS were prepared using fluorescent-labeled nonsensical siRNA (FAM-siNC) instead of siATG7. Rat stellate cells HSC-T6 were seeded in 6-well plates overnight until the cells reached 80-90% coverage, replaced with serum-free medium, and incubated with Lip/FAM-siNC/CK1, lip/FAM-siNC/CS for 4h at a FAM-siNC dosing concentration of 0.5 μg/mL. The cells were collected by digestion, washed three times with PBS and examined by flow cytometry.
As shown in (A), (B) and (C) in the attached figure 2, the measurement results show that the modification of CK1 and CS can greatly improve the uptake of FAM-siNC/Lip. Under the condition that the C/N ratio is 0.6-3.6:1, when the C/N ratio is greater than or equal to 1:1, the relative uptake rate of CS reaches 100%, and the relative uptake rate of FAM-siNC/Lip is only 3.6%, which shows that the CD44 targeting effect mediated by chondroitin sulfate greatly promotes uptake. For CK1, uptake gradually increased as the C/N ratio increased. The C/N ratio was increased from 0.6:1 to 2.8:1, and the relative uptake was increased stepwise from 37.2% to 100.4%.
Experimental example 2
Silencing effect of different C/N ratios Lip/siATG7/CK1 on ATG7 protein was determined by WB (immunoblotting experiment):
TGF-. Beta.1 is one of the major stimulators of hepatic stellate cell activation. In vitro experiments, we activated hepatic stellate cells by TGF- β1 stimulation to enhance autophagy levels, with significant upregulation of ATG7 expression. Based on cellular uptake, C/N1:1, 1.8:1 and 2.8:1 were selected for in vitro gene silencing studies.
The specific method comprises the following steps: HSC-T6 was seeded in 6-well plates overnight until the cells reached 80-90% coverage. It was incubated with Lip/siATG7/CK1 at C/N ratios of 1:1, 1.8:1 and 2.8:1, respectively, for 4h, fresh medium was replaced, incubated with 10. Mu.g/mL TGF-. Beta.for 48h, and ATG7 expression was detected. A commercially available transfection reagent GP-Transfect-Mate (hereinafter referred to as Mate) (Ji Ma gene, shanghai, china) was used as a positive control (FIG. 3D). Protein expression gradually decreases with increasing C/N ratio, which may be an effect of cellular uptake. At a C/N ratio of 2.8:1, the Lip/siATG7/CK gene silencing effect is equivalent to that of a positive control Mate. In summary, a C/N ratio of 2.8:1 was chosen as the optimal C/N ratio for subsequent study. At this time, the particle size potentials of the Lip/siATG7/CS and the Lip/siATG7/CK are 228.5+ -0.2 nm/-37.6+ -0.2 mV, 222.6+ -0.2 nm/-38.2+ -0.3 mV, respectively, and the PDI is less than 0.2. The experimental results are shown in (D) of the attached figure 3 of the specification. In addition, we synthesized CK2 with higher KDEL modification degree (modification degree is 48%) as a control, and the ingestion experiment result shows that the Lip/FAM-siNC/CK1 and Lip/FAM-siNC/CS are highest in ingestion, no significant difference exists, the ingestion of Lip/FAM-siNC/CK2 is only 29% of Lip/FAM-siNC/CK1, the influence of the polypeptide modification degree on cell targeting is verified again, and the experiment result is shown in (E) of an attached drawing 3 of the specification.
Experimental example 3
The endocytic mechanism of the nanoparticle was determined: the specific inhibitors of the three major endocytic pathway, chlorpromazine, methyl beta cyclodextrin, amiloride, were used to inhibit clathrin-mediated endocytosis, and caveolin-mediated endocytosis and megacytosis, respectively. As shown in the attached figure 3 (F), the experimental result shows that the methyl beta cyclodextrin has significant downregulation on the uptake of Lip/FAM-NC/CK1, amiloride and chlorpromazine do not influence the uptake basically, and the Lip/FAMsiNC/CS and Lip/FAM-NC/CK1 enter cells mainly through the endocytosis pathway mediated by the pit protein. Whereas uptake of Lip/FAMsiNC/CK2 was reduced by amiloride and chlorpromazine and not affected by methyl- β -cyclodextrin, indicating that Lip/FAMsiNC/CK2 enters the cell mainly through clathrin-mediated endocytosis and megapinocytosis. The above results indicate that excessive polypeptide modification not only affects targeting ability, but also alters cellular uptake mechanisms.
Experimental example 4
Measuring gene and protein expression after RNA interference, and examining gene silencing efficiency:
the specific experimental method comprises the following steps: HSC-T6 was seeded in 12-well plates overnight until the cells reached 80-90% coverage. Cells were incubated with nanocomposites such as Lip/siATG7/CK1 for 6h, and finally replaced with 10ng/mL TGF-. Beta.1 for 36h to detect mRNA levels using q-PCR, or for 48h to detect protein expression.
As shown in the attached drawing 4 (A-E), the Lip/siATG7/CK1 reduces the cell ATG mRNA to 42%, which is better than Lip/siATG7/CS and Lip/siATG7/CK2, and the expression is more obvious at the protein level. In normal liver, hepatic stellate cells serve as the primary vitamin a storage repository throughout the body, storing them in the form of retinol lipid in perinuclear fat droplets. During liver injury, hepatic stellate cells differentiate or activate into myofibroblast phenotypes (MFBs) with high proliferation, high contractility, fibrosis, high chemotaxis, becoming a major source of fibrosis. alpha-SMA is an indicator of astrocyte activation. Extracellular matrices such as type I Collagen (Collagen I) are produced after the activation of astrocytes, so that the generation of fibrosis Lip/siATG7/CK1 significantly reduces the protein expression of ATG7, collagen I and a-SMA to a pre-activation level. The experiment shows that Lip/siATG7/CK1 can perform effective gene interference and has excellent in-vitro anti-fibrosis effect.
Experimental example 5
Fat droplet metabolism experiment and scratch experiment:
the specific method comprises the following steps: 20mg/mL of palmitic acid solution and 14mg/mL of retinol solution were prepared using ethanol, 1mM BSA solution (about 66 mg/mL) was prepared using ultrapure water, and 40. Mu.L of Palmitic Acid (PA) and 6.4. Mu.L of retinol solution were added dropwise to 800. Mu.L of BSA solution under vortex. The above solution was then mixed well with the medium at 1:19 and incubated with the cells for 24 hours, i.e., to form vitamin A fat droplets (retinol about 20. Mu.M, PA 100. Mu.M) in HSC-T6 cells. After medium replacement, PBS, lip/siATG7/CS, lip/siATG7/CK1, lip/siATG7/CK2 were added and incubated with cells for 6 hours, commercial formulation mate was used as positive control, and after medium replacement with 10ng/mL TGF-. Beta.for 48 hours, fat droplets were observed under a microscope for metabolism using oil red O staining.
As a result of the experiment, as shown in FIG. 5 (C), the fat droplets were observed to disappear after the TGF-. Beta.activated astrocytes. Lip/siATG7/CK1 inhibits the disappearance of fat droplets, and the inhibition effect is stronger than that of Lip/siATG7/CS and Lip/siATG7/CK2.
The migration ability of the cells was evaluated by a scratch test:
the specific method comprises the following steps: HSC-T6 cells were seeded in 12-well plates and photographed using a microscope using 200. Mu.L gun heads to draw a straight line when cell density reached 90% or higher. Cells were incubated with nanocomposites such as Lip/siATG7/CK1 for 6h, and finally replaced with 10ng/mL TGF-. Beta.1 for 48h, and then photographed using a microscope to record scratch healing.
As shown in the attached figure 5 (B), the Lip/siATG7/CK1 can significantly inhibit the healing of HSC-T6 scratches, and has stronger effect than Lip/siATG7/CS and Lip/siATG7/CK2.
The above experiments demonstrate that Lip/siATG7/CK1 inhibits hepatic stellate cell activation by lipid-phagocytosis inhibition, thereby inhibiting fibrosis progression.
Experimental example 6
Organelle localization of the nanoparticle: the golgi is a modification site for protein glycosylation, and there are a large number of Glycosyltransferases (GTs), glycosidases, and the like. GalNAc groups on cartilage sulphate can be targeted to the Golgi apparatus via GalNAc-Ts.
The specific method comprises the following steps: the distribution of Lip/FAM-siNC/CK1 in the organelles was examined using FAM-siNC as a fluorescently labeled siRNA. After incubating Lip/FAM-siNC/CK1, lip/FAM-siNC/CK2 with cells for 2 hours, the cells were washed three times with PBS, and finally stained with the responsive organelle dyes, respectively, and observed with a confocal microscope. The experimental results are shown in the specification and the attached figure 6 (A), wherein Lip/FAM-siNC/CS is co-located with the Golgi apparatus. Lip/FAM-siNC/CK1 was predominantly distributed in the endoplasmic reticulum, and none of the formulations of each group co-localized with lysosomes.
Experimental example 7
Using confocal microscopy, the release of genes in cells was observed by fluorescence resonance energy transfer experiments (FRET): FAM and Cy3 were used as FRET donor and acceptor, respectively, and the same amount of FAM-sinC was co-supported in Cy3-sinC in liposome to form complex Lip/FAM-sinC/Cy3-sinC/CK1 with CK1.
The specific experimental steps are as follows: FAM-siNC and Cy3-siNC are selected as a donor and an acceptor of FRET effect respectively, equal amounts of FAM-siNC and Cy3-siNC are mixed and then form a complex with cationic liposome, then Lip/FAM-siNC/Cy3-siNC/CK1 is formed with CK1, in addition, nanoparticle Lip/FAM-siNC/CK1, lip/Cy3-siNC/CK1 which respectively contain FAM-siNC or Cy3-siNC and a mixed group Lip/FAM-siNC/CK1 of two nanoparticles are arranged as a control, after 4h, 8h and 24h of administration, PBS is used for washing three times, then 4% of polyformic acid is used for fixing 10min, DAPI is used for dyeing cell nuclei for 5min, and after chip sealing, anti-fluorescence quenching agent is added, and confocal microscopy is used for observation.
The experimental results are shown in the accompanying figure 6 (B) of the specification: before release, an emitted light signal (FRET) of Cy3 will appear upon excitation by the excitation light wavelength of FAM; once the gene is released, the FRET signal disappears. 4h and 8h, we observed strong FRET signals, indicating that the nanoparticle maintained a complete structure before 8. At 24h, most of the FRET signal disappeared, indicating that the gene is being slowly released. Since accumulation of Lip/Cy5-sinC/CK1 has been observed in the endoplasmic reticulum at 2h, this suggests that Lip/Cy5-sinC/CK1 is transported to the endoplasmic reticulum in the form of nanoparticles rather than diffusing to the endoplasmic reticulum in the form of episomes.
Experimental example 8
Transport mechanism investigation:
the specific method comprises the following steps: the procedure was as in experimental example 6, with GCA incubated with cells for half an hour in advance before the cells were incubated with Lip/FAM-siNC/CK 1.
The experimental results are shown in the accompanying figure 7 (A-B) of the specification: GCA is a specific inhibitor of copi and after GCA treatment of cells we observed a decrease in accumulation of Lip/FAM-siNC/CK1 in the endoplasmic reticulum, whereas co-localization occurred in the golgi apparatus, indicating that Lip/FAM-siNC/CK1 is targeted to the endoplasmic reticulum by copi vesicle-mediated transport through the golgi apparatus.
Examining the effect of the golgi-endoplasmic reticulum pathway on gene interference effects: the specific method comprises the following steps: HSC-T6 was seeded in 12-well plates overnight until the cells reached 80-90% coverage. Incubating 20. Mu.g/mL GalNAc or 10. Mu.M GCA with cells for half an hour, replacing the culture medium, incubating the cells with a nanocomposite such as Lip/siATG7/CK1 for 6 hours, and finally replacing 10ng/mL TGF-beta 1 for 36 hours, and detecting mRNA level by q-PCR
The experimental results are shown in the accompanying figure 7 (C) of the specification: galNAc is a competitive inhibitor of chondroitin sulfate targeting golgi. GalNAc and GCA inhibited the gene silencing efficiency of Lip/siATG7/CK1 by 91% and 61.9%, respectively, indicating that Golgi targeting and Golgi-endoplasmic reticulum transport are critical for Lip/siATG7/CK1 to exert effective gene interference.
Experimental example 9
Establishing a carbon tetrachloride-induced liver fibrosis model, and determining the in-vivo targeting of nanoparticles:
the specific method for establishing the liver fibrosis model comprises the following steps: c57 male mice, 18-20g, are selected, carbon tetrachloride is dissolved in olive oil to form 10% solution, the solution is injected intraperitoneally at a dosage of 2mL/kg, the injection is carried out once every other day, and liver fibrosis is formed four weeks after continuous injection.
The detection method of the in vivo targeting comprises the following steps: lip/Cy5-siNC/CK1, lip/Cy5-siNC/CS, lip/Cy5-siNC/CK2 were prepared, and the final Cy5-siNC concentration was 60. Mu.g/mL, 0.2mL (0.6 mg/kg) was administered to each mouse, 4 mice per group. Taking pictures through a living animal imager 2h and 4h after administration, killing 2 mice at the neck of each of 4h and 6h, respectively, and taking isolated viscera and taking pictures after heart perfusion with PBS and 4% chloral hydrate. The receptor-blocked group was pre-injected with 10mg/kg chondroitin sulfate 1h before dosing.
The experimental results are shown in the attached figure 8 of the specification: first, cy5-siNC is rapidly metabolized by the kidney and has little accumulation in the liver. Lip/Cy5-siNC/CS and Lip/Cy5-siNC/CK1 have the strongest accumulation in the liver. Meanwhile, the pre-blocking significance of the chondroitin sulfate reduces the accumulation of Lip/Cy5-siNC/CK1 in the liver, which indicates that the in-vivo targeting effect of Lip/Cy5-siNC/CK1 is mediated by the related targeting of the chondroitin sulfate. Accumulation of Lip/Cy5-siNC/CK1 was significantly reduced in normal liver compared to fibrotic liver, probably because fibrotic liver significantly upregulated the expression of the specific receptor CD44 of chondroitin sulfate. The fibrous liver targeting of Lip/Cy5-siNC/CK2 was inferior to Lip/Cy5-siNC/CK1, consistent with in vitro experiments. Hepatic stellate cells, hepatic sinus endothelial cells, and hepatic macrophages are three non-parenchymal cells of the liver. Autophagy has different directional effects on fibrosis in different cells, on one hand promoting activation of hepatic stellate cells, and on the other hand generating anti-fibrosis signals in hepatocytes, macrophages, endothelial cells by liver protection and anti-inflammatory effects. Specific cell localization is a prerequisite for anti-fibrosis therapies targeting autophagy.
We analyzed liver sections by immunofluorescence to further determine the cellular localization of nanoparticles in the liver:
the experimental results are shown in the attached figure 8 of the specification: the Lip/Cy5-siNC/CK1 is consistent with hepatic stellate cell distribution, while the Lip/Cy5-siNC/CK2 is mainly captured by hepatic sinus endothelial cells and partially consistent with the stellate cell distribution. According to the experimental results, the modification degree of the polypeptide greatly influences the liver targeting, cell positioning and intracellular fate of the nanoparticles, thereby influencing the gene silencing efficiency.
Experimental example 10
Investigation of anti-hepatic fibrosis effect in vivo:
since autophagy is involved in the activation process of astrocytes, a dosing regimen as shown in fig. 9 (a) is adopted, and the specific method is as follows: c57 male mice, 18-20g, are selected, carbon tetrachloride is dissolved in olive oil to form 10% solution, and the solution is injected intraperitoneally at a dosage of 2mL/kg once every other day, and the injection is continued for four weeks. The last three carbon tetrachloride period is not administered, and the last day of carbon tetrachloride is followed by beginning tail vein injection of PBS, lip/siATG7/CS, lip/siNC/CK, lip/siATG7/CK, siATG dosage is injected once every 2 days at 1mg/kg, blood is taken through the eye socket after the last administration is completed for 48 hours, plasma is collected by centrifugation, and plasma AST and ALT concentration are measured by ELISA. Livers were collected after euthanasia of mice and liver fibrosis levels were assessed by Masson staining and immunohistochemistry.
As can be seen in FIGS. 9 (B-F), the normal liver showed a smooth surface, whereas the fibrotic liver surface was rough, and liver surface roughness was improved in mice treated with Lip/siATG7/CK 1. Serum AST and ALT levels can reflect to some extent the extent of liver injury. Both Lip/siATG7/CK1 and Lip/siATG7/CS significantly inhibited serum AST and ALT levels. After liver sections were subjected to immunohistochemical staining and Masson staining. After liver fibrosis, type I collagen and fibers are generated in a large quantity around liver sinuses, and the generation of fibrosis is obviously reduced by Lip/siATG7/CS and Lip/siATG7/CK1, and Lip/siATG7/CK1 shows the optimal in vivo anti-fibrosis effect, so that the liver fibrosis treatment method has the advantages of targeting activated hepatic stellate cells and excellent gene silencing efficiency.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (7)
1. A polysaccharide-polypeptide conjugate for high efficiency delivery of siRNA, characterized by:
the polysaccharide-polypeptide conjugate is KDEL polypeptide grafted chondroitin sulfate, and has the structural formula:
wherein a and b are positive integers more than or equal to 1, R is SO 3 - ；
The molar ratio of the chondroitin sulfate to the KDEL polypeptide is 1:0.2-1;
the polysaccharide-polypeptide conjugate is used for delivering siRNA;
the way of carrying and delivering siRNA by the polysaccharide-polypeptide conjugate comprises the following steps:
1) Preparing a cationic gene compression vector;
2) Incubating the siRNA with a cationic gene compression carrier to form a CV/siRNA complex;
3) The polypeptide-polysaccharide conjugate is dissolved in PBS, and then CV/siRNA complex is dripped into the solution to form CV/siRNA/CK.
2. A method of preparing a polysaccharide-polypeptide conjugate of claim 1, comprising the steps of:
chondroitin sulfate, EDC and NHS are dissolved in PBS buffer solution for activation, then KDEL polypeptide is added, the pH is adjusted to 7-8, and the KDEL polypeptide is grafted on the chondroitin sulfate through amide reaction.
3. The preparation method according to claim 2, characterized in that: the activation temperature is 10-45 ℃; and/or the temperature of the amide reaction is 10-45 ℃, and the time of the amide reaction is 0.5-72h.
4. The polysaccharide-polypeptide conjugate of claim 1, wherein: the siRNA includes siATG7.
5. The polysaccharide-polypeptide conjugate of claim 1, wherein: when the polysaccharide-polypeptide conjugate is used to deliver siATG7, the KDEL has a grafting ratio of 5-90%.
6. The polysaccharide-polypeptide conjugate of claim 1, wherein: in step 1), the cationic gene compression vector comprises cationic liposome, polyethyleneimine/PEI, polymethacrylate, DGL, polyamide-amine dendrimer, polylysine, chitosan or protamine.
7. The polysaccharide-polypeptide conjugate of claim 1, wherein: in the step 2), the molar ratio of N of the cationic gene compression vector to P in the siRNA is greater than 4:1;
and/or, in step 3), the molar ratio of the carboxyl group on the chondroitin sulfate backbone of the polysaccharide-polypeptide conjugate to the N in the cationic gene compression carrier is 0.6-3.6:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310133398.5A CN116236584B (en) | 2023-02-20 | 2023-02-20 | Polysaccharide-polypeptide conjugate for high-efficiency siRNA delivery |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310133398.5A CN116236584B (en) | 2023-02-20 | 2023-02-20 | Polysaccharide-polypeptide conjugate for high-efficiency siRNA delivery |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116236584A CN116236584A (en) | 2023-06-09 |
| CN116236584B true CN116236584B (en) | 2024-03-22 |
Family
ID=86629135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310133398.5A Active CN116236584B (en) | 2023-02-20 | 2023-02-20 | Polysaccharide-polypeptide conjugate for high-efficiency siRNA delivery |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116236584B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1272882A (en) * | 1997-07-18 | 2000-11-08 | 居里研究所 | Chimeric polypeptide comprising fragment B of shiga toxin and peptides of therapeutic interest |
| TW201742958A (en) * | 2016-06-11 | 2017-12-16 | 中央研究院 | High-throughput screening of functional antibody fragments, immunoconjugate comprising the same, and adaptor-drug conjugate for screening |
| CN111328287A (en) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | Novel nucleic acid molecules |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1229045A1 (en) * | 2001-02-01 | 2002-08-07 | Institut Curie | Universal carrier for targeting molecules to Gb3 receptor expressing cells |
| US9487773B2 (en) * | 2013-03-01 | 2016-11-08 | Technophage, Investigacao E Desenvolvimento Em Biotecnologia, Sa | Cell-based methods for coupling protein interactions and binding molecule selection |
-
2023
- 2023-02-20 CN CN202310133398.5A patent/CN116236584B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1272882A (en) * | 1997-07-18 | 2000-11-08 | 居里研究所 | Chimeric polypeptide comprising fragment B of shiga toxin and peptides of therapeutic interest |
| TW201742958A (en) * | 2016-06-11 | 2017-12-16 | 中央研究院 | High-throughput screening of functional antibody fragments, immunoconjugate comprising the same, and adaptor-drug conjugate for screening |
| CN111328287A (en) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | Novel nucleic acid molecules |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116236584A (en) | 2023-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhao et al. | Exosome-mediated siRNA delivery to suppress postoperative breast cancer metastasis | |
| Muddineti et al. | Cholesterol-grafted chitosan micelles as a nanocarrier system for drug-siRNA co-delivery to the lung cancer cells | |
| Sun et al. | RETRACTED ARTICLE: siRNA-loaded poly (histidine-arginine) 6-modified chitosan nanoparticle with enhanced cell-penetrating and endosomal escape capacities for suppressing breast tumor metastasis | |
| Chang et al. | Efficient gene transfection by histidine-modified chitosan through enhancement of endosomal escape | |
| Gao et al. | Arginine-chitosan/DNA self-assemble nanoparticles for gene delivery: In vitro characteristics and transfection efficiency | |
| Mann et al. | Thioaptamer conjugated liposomes for tumor vasculature targeting | |
| Morris et al. | Folate mediated histidine derivative of quaternised chitosan as a gene delivery vector | |
| Zheng et al. | Self-assembled dual fluorescence nanoparticles for CD44-targeted delivery of anti-miR-27a in liver cancer theranostics | |
| Yang et al. | ROS-responsive fluorinated polyethyleneimine vector to co-deliver shMTHFD2 and shGPX4 plasmids induces ferroptosis and apoptosis for cancer therapy | |
| Nuñez-Rivera et al. | Brome mosaic virus-like particles as siRNA nanocarriers for biomedical purposes | |
| Zhao et al. | Chitosan derived glycolipid nanoparticles for magnetic resonance imaging guided photodynamic therapy of cancer | |
| Chu et al. | Biocompatible polyethylenimine-graft-dextran catiomer for highly efficient gene delivery assisted by a nuclear targeting ligand | |
| Wang et al. | Dual‐targeting heparin‐based nanoparticles that re‐assemble in blood for glioma therapy through both anti‐proliferation and anti‐angiogenesis | |
| Han et al. | pH-Sensitive tumor-targeted hyperbranched system based on glycogen nanoparticles for liver cancer therapy | |
| Wang et al. | Tumor-selective lipopolyplex encapsulated small active RNA hampers colorectal cancer growth in vitro and in orthotopic murine | |
| CN107184987B (en) | Lipoic acid modified targeted integrin alpha v beta 3 nano-polypeptide carrier and preparation method and application thereof | |
| WO2007086923A2 (en) | Controlled and sustained gene transfer mediated by thiol-modified polymers | |
| Song et al. | Erythrocyte-biomimetic nanosystems to improve antitumor effects of paclitaxel on epithelial cancers | |
| CN109381705A (en) | Reversible crosslink Biodegradable polymer vesicles and preparation method thereof with asymmetric membrane structure | |
| WO2015125147A9 (en) | Anionic polyplexes for use in the delivery of nucleic acids | |
| Zhou et al. | Tailoring the supramolecular structure of guanidinylated pullulan toward enhanced genetic photodynamic therapy | |
| Park et al. | Polyethylenimine grafted-chitosan based Gambogic acid copolymers for targeting cancer cells overexpressing transferrin receptors | |
| Fang et al. | A cancer cell membrane coated nanoparticles-based gene delivery system for enhancing cancer therapy | |
| Tang et al. | Functionalized PAMAM-Based system for targeted delivery of miR-205 and 5-fluorouracil in breast cancer | |
| CN116236584B (en) | Polysaccharide-polypeptide conjugate for high-efficiency siRNA delivery |
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 |