CN106188555B - A kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system and preparation method and application - Google Patents
A kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system and preparation method and application Download PDFInfo
- Publication number
- CN106188555B CN106188555B CN201610559302.1A CN201610559302A CN106188555B CN 106188555 B CN106188555 B CN 106188555B CN 201610559302 A CN201610559302 A CN 201610559302A CN 106188555 B CN106188555 B CN 106188555B
- Authority
- CN
- China
- Prior art keywords
- sirna
- acid
- delivery system
- poly
- preparation
- 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 84
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 230000004043 responsiveness Effects 0.000 title claims abstract description 15
- 230000008685 targeting Effects 0.000 title claims abstract description 10
- 229920000642 polymer Polymers 0.000 claims abstract description 54
- 239000002253 acid Substances 0.000 claims abstract description 34
- 150000001875 compounds Chemical class 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 16
- 239000002105 nanoparticle Substances 0.000 claims abstract description 16
- -1 poly- (diisopropylaminoethyl) ethyl Chemical group 0.000 claims abstract description 14
- 229940079593 drug Drugs 0.000 claims abstract description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 10
- 241000432824 Asparagus densiflorus Species 0.000 claims abstract description 8
- 125000002252 acyl group Chemical group 0.000 claims abstract description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 6
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 15
- 229920001510 poly[2-(diisopropylamino)ethyl methacrylate] polymer Polymers 0.000 claims description 14
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims description 9
- 201000011510 cancer Diseases 0.000 claims description 9
- NJSRYBIBUXBNSW-VIFPVBQESA-N (3s)-3-azaniumyl-4-oxo-4-phenylmethoxybutanoate Chemical compound [O-]C(=O)C[C@H]([NH3+])C(=O)OCC1=CC=CC=C1 NJSRYBIBUXBNSW-VIFPVBQESA-N 0.000 claims description 7
- 150000003512 tertiary amines Chemical class 0.000 claims description 7
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 claims description 6
- 235000003704 aspartic acid Nutrition 0.000 claims description 6
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 5
- 238000003745 diagnosis Methods 0.000 claims description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 4
- 238000006116 polymerization reaction Methods 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 3
- 239000005864 Sulphur Substances 0.000 claims description 3
- 238000005915 ammonolysis reaction Methods 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 235000019445 benzyl alcohol Nutrition 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 claims description 3
- 150000008065 acid anhydrides Chemical group 0.000 claims description 2
- 238000011938 amidation process Methods 0.000 claims description 2
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000003054 catalyst Substances 0.000 claims description 2
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 claims description 2
- SUPCQIBBMFXVTL-UHFFFAOYSA-N ethyl 2-methylprop-2-enoate Chemical compound CCOC(=O)C(C)=C SUPCQIBBMFXVTL-UHFFFAOYSA-N 0.000 claims description 2
- 229960003151 mercaptamine Drugs 0.000 claims description 2
- 229940068917 polyethylene glycols Drugs 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 238000007151 ring opening polymerisation reaction Methods 0.000 claims description 2
- 229920000428 triblock copolymer Polymers 0.000 claims description 2
- JIGUQPWFLRLWPJ-UHFFFAOYSA-N Ethyl acrylate Chemical compound CCOC(=O)C=C JIGUQPWFLRLWPJ-UHFFFAOYSA-N 0.000 claims 2
- 125000006202 diisopropylaminoethyl group Chemical group [H]C([H])([H])C([H])(N(C([H])([H])C([H])([H])*)C([H])(C([H])([H])[H])C([H])([H])[H])C([H])([H])[H] 0.000 claims 2
- PNJWIWWMYCMZRO-UHFFFAOYSA-N pent‐4‐en‐2‐one Natural products CC(=O)CC=C PNJWIWWMYCMZRO-UHFFFAOYSA-N 0.000 claims 2
- WZZBNLYBHUDSHF-DHLKQENFSA-N 1-[(3s,4s)-4-[8-(2-chloro-4-pyrimidin-2-yloxyphenyl)-7-fluoro-2-methylimidazo[4,5-c]quinolin-1-yl]-3-fluoropiperidin-1-yl]-2-hydroxyethanone Chemical compound CC1=NC2=CN=C3C=C(F)C(C=4C(=CC(OC=5N=CC=CN=5)=CC=4)Cl)=CC3=C2N1[C@H]1CCN(C(=O)CO)C[C@@H]1F WZZBNLYBHUDSHF-DHLKQENFSA-N 0.000 claims 1
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 claims 1
- 239000003999 initiator Substances 0.000 claims 1
- 238000001476 gene delivery Methods 0.000 abstract description 2
- 238000003759 clinical diagnosis Methods 0.000 abstract 1
- 229920001577 copolymer Polymers 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
- 230000005611 electricity Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 230000005588 protonation Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 229920002873 Polyethylenimine Polymers 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 5
- 150000001768 cations Chemical class 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 229960005261 aspartic acid Drugs 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 230000005595 deprotonation Effects 0.000 description 4
- 238000010537 deprotonation reaction Methods 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108010039918 Polylysine Proteins 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 3
- 230000004700 cellular uptake Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 230000012202 endocytosis Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 230000002132 lysosomal effect Effects 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229920000656 polylysine Polymers 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010025 steaming Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 2,3-dimethylbutane Chemical group CC(C)C(C)C ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 0.000 description 1
- HIYNVGBAVGTAPV-UHFFFAOYSA-N 5-[di(propan-2-yl)amino]-2-methylpent-2-enoic acid Chemical compound CC(C)N(C(C)C)CCC=C(C)C(O)=O HIYNVGBAVGTAPV-UHFFFAOYSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 241000555268 Dendroides Species 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000006392 deoxygenation reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 206010016766 flatulence Diseases 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229920000867 polyelectrolyte Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000036326 tumor accumulation Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
- C08G81/02—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers at least one of the polymers being obtained by reactions involving only carbon-to-carbon unsaturated bonds
- C08G81/024—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G
- C08G81/028—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G containing polyamide sequences
-
- 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
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/14—Lactams
- C08G69/16—Preparatory processes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention particularly discloses a kind of tumour intelligence targeting and environment double responsiveness siRNA delivery system and preparation method and application.The siRNA delivery system is that siRNA concentration is compound in nanoparticle core by acid-sensitive amphiphilic triblock polymer, and subsurface stratum forms intermolecular disulfide bond realization to the protection of siRNA by PAsp (MEA) and responds siRNA release in reproducibility endochylema.The acid-sensitive amphiphilic triblock polymer is made of polyethylene glycol section-three sections of copolymers of acid-sensitive section of interlude-, and interlude is poly- asparagus fern acyl mercaptoethylmaine, and acid-sensitive section is poly- (diisopropylaminoethyl) ethyl methacrylate.SiRNA delivery system can intelligently target siRNA Nano medication using preparation tumour, lower than degree of dependence to N/P, it can be achieved that target spot quickly and completely discharges siRNA.A new thinking is provided for gene delivery system, is of great significance to the clinical diagnosis and therapeutic agent of preparation tumour.
Description
Technical field
The present invention relates to polymer chemistry and biomedical engineering field, intelligently target more particularly, to a kind of tumour
With environment double responsiveness siRNA delivery system and preparation method and application.
Background technique
Malignant tumour, also known as cancer have become the major disease for threatening human health and life.To the pre- of malignant tumour
Anti-, diagnosing and treating is always the problem of people earnestly pay close attention to, the attention of progress and national governments recently as science and technology,
Although achieving great progress in this aspect, various treatment methods all encounter different difficulties, it is difficult to while realizing height
Effect, less toxic side effect and high specific treatment.
In recent years, RNA interference provides important reference means for oncotherapy because its is selectively targeted, and shows height
The previous experiments room Gene silencing efficacy of effect.However, because the transmission problem of siRNA, clinical application still suffers from great
Challenge.It is well known that negative electrical charge of traditional siRNA molecule because of its biggish molecular weight and institute's band itself, it is extremely difficult to pass through cell
Film realizes endocytosis；Importantly, lesions position generally can not be reached because of the characteristic that it is easily degraded by nuclease；And even if
Local administration and enter cell, the possibility that the degradation of lysosomal enzyme has also blocked it to play a role to endochylema.This is allowed for
Its direct clinical application is almost impossible.Therefore, the siRNA Transmission system of stability and high efficiency is to realize tumor RNA interference treatment
It is crucial.The polymer cation carrier shown one's talent from numerous siRNA transmission systems has following advantage: lower immunogene
Property and toxicity (compared with viral vectors), the controllability of partial size, the introducing of multifunction easy to accomplish and targeted molecular.So far, compared with
Mature polymer cation carrier includes: polyethyleneimine (PEI), polylysine (PLL), imidazoles quasi polymer, chitosan
And cation dendroid macromolecule.Cationic polymer is acted on by polyelectrolyte and the siRNA of anion is concentrated and is encapsulated, group
Dress up the compound of nano-scale.SiRNA is effectively protected in this way, and enzyme is avoided to degrade, at the same the nitrogen of enough big polymer and
Under the molar ratio (N/P ratio) of the phosphorus of siRNA, i.e. when the cation of polymer is more than needed, nano-complex surface can be positively charged, can solve
The problem of certainly naked siRNA difficulty is absorbed by cell.However, this common nano-complex, since there are non-specific cell suctions
Receive, in the short and easy absorption blood of blood circulation time Anionic Protein and the problems such as assemble, vivo applications are limited significantly.On the contrary
Ground, the negative electricity nano-complex (low N/P is than compound) that conventional method obtains are often difficult by cellular uptake due to the negative electricity of surface.More
Have notably, positive electricity deficiency generally means that siRNA fails to be fully protected, this allows for siRNA and is easily degraded by enzymes and is not achieved
Convey purpose.In addition, it is this it is not exclusively compound be easy to cause biggish nano-scale, easily swallowed by reticuloendothelial system (RES),
It not easily passs through enhancing infiltration retention effect (EPR) and realizes tumor accumulation.Therefore, effective delivery vehicles system should have both above-mentioned two
The advantages of kind of nanoparticle and the shortcomings that them can be evaded.
In order to achieve the above objectives, a kind of cancer target strategy of " surface potential reversion " is suggested and is applied to gene
Transmission.Specifically, using poly- (polyethylene glycol-histidine-polyethylene glycol-glutamic acid (poly (PEG-His-PEG-Glu)) copolymerization
Object and PEI25kIt is compound under specific ratio with gene, it obtains (pH 7.4) surface potential under the conditions of physiological environment and is negative
Nanoparticle, the nanoparticle can stablize circulation in blood for a long time, and after reaching tumor locus, due to the microenvironment of weak acid
The strong proton buffering effect of (pH 6.8) and PEI, nanoparticle surface meeting current potential is changed into positive electricity, can promote cell endocytosis, real
The specific targeting of existing tumour.However, these obvious disadvantages restrict further applying for the system.Firstly, the complex systems
Surface potential reverse speed characterisstic to proportions require it is very stringent, be just able to achieve compound in very limited proportional region
The reversion of surface potential, it is difficult to control；Secondly, PEI discharges genomic medicine, process as siRNA carrier, by the slow effect of proton
Slow and final burst size is too low, causes siRNA concentration used higher and wastes serious；Furthermore the molecular weight branched PEI of 25kDa
Use, toxicity problem is not suitable for long-term treatment.It is defeated therefore, it is necessary to develop a kind of new gene on the basis of above-mentioned strategy
System is sent to solve the problems, such as above various.
Summary of the invention
The present invention is in order to overcome existing genophore to be difficult to realize internal long circulating simultaneously and easily absorbed by tumour cell
Defect, provides a kind of acid-sensitive amphiphilic triblock polymer, and siRNA can be concentrated and be compound in nanometer by the triblock polymer
In particle core.
Another object of the present invention is to provide the preparation methods of above-mentioned triblock polymer.
Another object of the present invention is to provide a kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system,
The delivery system is the siRNA Nano medication for being provided simultaneously with above-mentioned two o'clock property, i.e. body-internal-circulation surface is negatively charged but in tumour
Tissue can voluntarily current potential invert, and realize that tumour intelligently targets.
Another object of the present invention is to provide a kind of tumours intelligently to target and environment double responsiveness siRNA delivery system
Preparation method.
Another object of the present invention is to provide the siRNA delivery systems in preparation cancer diagnosis drug and/or treatment
Application in drug.
Above-mentioned purpose of the invention is achieved by the following technical programs.
A kind of acid-sensitive amphiphilic triblock polymer, the polymer is by polyethylene glycol section-acid-sensitive section of interlude-three block
Copolymer is constituted, and the interlude is poly- asparagus fern acyl mercaptoethylmaine, and acid-sensitive section is poly- (diisopropylaminoethyl) methacrylic acid second
Ester, the pKa of poly- (diisopropylaminoethyl) ethyl methacrylate are 6.3~6.8.
PEG in acid-sensitive amphiphilic triblock polymer can improve stability and biocompatibility；Interlude is with poly- asparagus fern
Propylhomoserin is main chain, degradable, and the introducing of sulfydryl can form intermolecular disulfide bond, improves stability；It is complete when acid-sensitive section of pH 5.0
Protonation, it can be achieved that siRNA load, 7.4 when complete deprotonation, it can be achieved that siRNA release.
Inventive point of the invention is acid-sensitive section of screening, due to acid-sensitive section of special pKa, makes the tumour intelligence finally obtained
It can target and environment double responsiveness siRNA delivery system substantially reduces the dependence of N/P ratio, and realize target spot quickly and completely
SiRNA release.Quickly and completely release siRNA needs acid-sensitive section of need to have following characteristics: there is stronger proton buffering effect,
It can guarantee the lysosome escape of siRNA；Need to have the very big matter of difference when from lysosomal pH (~5.0) to endochylema pH (~7.4)
Sub-ization degree is just able to achieve the efficient release of siRNA preferably from complete protonation to complete deprotonation in this way.Poly- (diisopropyl
Base amino) ethyl methacrylate (PDPA) pKa 6.3~6.8, above-mentioned requirements can be met.
It is highly preferred that the pKa of acid-sensitive section of poly- (diisopropylaminoethyl) ethyl methacrylate is 6.4.
Preferably, the PEG molecular weight that the acid-sensitive amphiphilic triblock polymer is selected is 1000~5000Da；Interlude
Cross-linked layer is too thin, bundlees loosely, too thick, is difficult to be opened in the cell, therefore, the poly- preferred molecule of asparagus fern acyl mercaptoethylmaine
Amount is 800~2000Da；Acid-sensitive section because in endochylema environment completely it is hydrophobic, triblock polymer is easily self-assembled into micella, if
It is to be assembled again by original nanoparticle disintegration, then siRNA discharges, and if not assembling again, need the expansion of its partial size enough
Greatly, therefore, the preferred molecular weight of poly- (diisopropylaminoethyl) ethyl methacrylate is 8000~12000Da.
It is highly preferred that the molecular weight of the polyethylene glycol section is 2000Da, the molecular weight of poly- asparagus fern acyl mercaptoethylmaine is
1000Da, the molecular weight of poly- (diisopropylaminoethyl) ethyl methacrylate are 10000Da.
The present invention protects acid-sensitive amphiphilic triblock polymer as described above to apply in preparation siRNA delivery system.
The present invention protects acid-sensitive amphiphilic triblock polymer as described above in preparation cancer diagnosis drug and/or treatment
Application in drug.
Preferably, the preparation method of acid-sensitive amphiphilic triblock polymer as described above, comprising the following steps:
S1. using aspartic acid and benzyl alcohol as raw material, using the concentrated sulfuric acid as catalyst, aspartic acid benzyl ester is prepared；
S2. using aspartic acid benzyl ester as raw material, it is slowly added to triphosgene, benzyloxycarbonyl group asparic acid is prepared in reflux
Acid anhydride (BLA-NCA)；
S3. with amino-polyethyleneglycols (PEG-NH2) make macromole evocating agent, cause benzyloxycarbonyl group aspartic acid acid anhydrides
(BLA-NCA) ring-opening polymerisation obtains PEG-PBLA-NH2；
S4. with 2- (dodecyl trithiocarbonic acid ester group) -2 Methylpropionic acid (DDAT) for chain-transferring agent, AIBN work causes
Agent causes the polymerization of monomer 2- (diisopropylaminoethyl) ethyl methacrylate (DPA), is taken off sulphur with excess AIBN after having reacted
It goes to obtain PDPA-COOH；
S5. pass through amidation process for PEG-PBLA-NH2It is docked with PDPA-COOH；
S6. it docks product with cysteamine with above-mentioned final product PEG-PAsp (MEA)-PDPA is obtained by ammonolysis reaction.
A kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system, the siRNA delivery system is by as above
SiRNA concentration is compound in nanoparticle core by the triblock polymer, and subsurface stratum forms intermolecular two sulphur by PAsp (MEA)
Key, which realizes the protection to siRNA and responds siRNA in reproducibility endochylema, to be discharged.The siRNA delivery system nanoparticle is because of it
Small particle and surface negative charge long-time stable can recycle in blood, be enriched to tumor locus by EPR effect.To tumour ring
Behind border, because of the change of microenvironment pH value, nanoparticle can show certain positive electricity because of the protonation of big quantity tertiary amine, be conducive to thin
Born of the same parents absorb.SiRNA delivery system of the invention solves existing genophore and is difficult to realize internal long circulating simultaneously and easily by tumour
The defect of cellular uptake, can recycle in vivo surface it is negatively charged but tumor tissues can voluntarily current potential invert, realize tumour medicine
Object intelligently targets.
It is highly preferred that the tumour intelligently targets and the preparation method of environment double responsiveness siRNA delivery system, including with
Lower step: triblock polymer PEG-PAsp (MEA)-PDPA is dissolved in the buffer solution that pH value is 5.0, and in current pH
Value is lower to press specific N/P with siRNA than compound 30min, under the conditions of same pH, bubble oxygen 30min is passed through into solution and allows Asia
The sulfydryl on surface layer is full cross-linked at disulfide bond, and the pH value of solution is finally adjusted to 7.4.
The present invention in order to avoid in negative electricity siRNA cannot introduce a kind of novel preparation side by fully compound
Formula.Specifically, the protonation of the compound siRNA in pH 5.0, acid-sensitive at this time section of tertiary amine are high, and polymer is containing more positive ionization
Son, energy siRNA are concentrated well in composite nano-granule daughter nucleus.Meanwhile reversible reduction sensitive layer is introduced in subsurface stratum, it will
SiRNA bundle protection is got up.Next, the pH value of solution is transferred to 7.4, acid-sensitive at this time section because of most of tertiary amine groups deprotonation
Cause hydrophobic, will form micellar structure, and due to the binding of subsurface stratum disulfide bond, change of size is little, and siRNA is more limited to
It in micella core, is better protected, which can show weaker negative electrical charge due to the presence of siRNA in core.This nanoparticle
Son because its small particle and surface negative charge can recycle for a long time in blood, considerably increase its be enriched to by EPR effect it is swollen
The probability of tumor.In pH 6.5, i.e., after siRNA delivery system reaches tumor environment, because of the change of microenvironment pH value, nanoparticle
Certain positive electricity can be shown because of the protonation of big quantity tertiary amine, be conducive to cell and absorb.
The present invention also protects tumour, and intelligently targeting and environment double responsiveness siRNA delivery system are preparing cancer diagnosis drug
And/or the application in therapeutic agent.
Compared with prior art, the invention has the following advantages:
The present invention provides a kind of tumours intelligently to target and environment double responsiveness siRNA delivery system and its preferred preparation side
Method, the siRNA delivery system are that siRNA concentration is compound in nanoparticle core by acid-sensitive amphiphilic triblock polymer,
Subsurface stratum forms intermolecular disulfide bond by PAsp (MEA) and realizes to the protection of siRNA and respond siRNA in reproducibility endochylema and release
It puts.SiRNA delivery system is in blood circulation, the stability with superelevation, substantially increases blood circulation time and tumour is rich
Collection；And it can efficiently be absorbed by tumour cell for the pH responsiveness of tumor microenvironment, GSH concentration is significantly when lysosome is escaped
It increases and the variation of pH value, triggering carrier is efficiently discharged because protonation reduces the transformation completed from hydrophilic to hydrophobic
SiRNA, or it is remarkably improved siRNA interference effect, siRNA delivery system of the invention solves existing genophore and is difficult to simultaneously
Realize internal long circulating and easily by tumour cell absorb defect, can recycle in vivo surface it is negatively charged but tumor tissues can
Voluntarily current potential inverts, and realizes that tumour medicine intelligently targets, provides a new thinking for gene delivery system.
Detailed description of the invention
Fig. 1 is the preparation of triblock polymer and intelligence targets and siRNA endochylema discharges schematic diagram.
Fig. 2 is the preparation route figure of triblock polymer PEG-PAsp (MEA)-PDPA.
Fig. 3 is the partial size potential diagram of triblock polymer at various ph values.
Fig. 4 is the gel blocking electrophoretogram of triblock polymer (N/P 18) after different condition is incubated for 1.5h.
Fig. 5 is the cytotoxicity test of triblock polymer.
Fig. 6 is cell under condition of different pH to the absorption efficiency of triblock polymer.
Specific embodiment
The present invention is described in further details with specific embodiment with reference to the accompanying drawings of the specification, but embodiment is not right
The present invention limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus are normal for the art
Advise reagent, method and apparatus.
The synthesis of 1 triblock polymer of embodiment
(1) synthesis of benzyloxycarbonyl group aspartic acid acid anhydrides (BLA-NCA), reaction mechanism and process are as follows:
First synthesis synthesis β-asparagine acid benzyl ester.500mL single port eggplant-shape bottle is taken, 100mL anhydrous ether is sequentially added,
12mL H2SO4(98%) and 120mL benzyl alcohol (1.2mol, 1.04g/mL, 108.06g/mol), ether is rotated after stirring.It stirs
It mixes down and the total 16g of L-Aspartic acid (0.12mol, 133.04g/mol) is added in three times.After being stirred to react 12h at room temperature, first it is added
The dilution of 95% ethyl alcohol of 240mL, then 60mL pyridine is added dropwise, white precipitate is precipitated.It is placed in refrigerator freezing and extracts solid out after overnight, it is pure
Water washing.Obtained crude product pure water recrystallizes twice at 80 DEG C, obtains pure aspartic acid benzyl ester.
BLA-NCA is synthesized by aspartic acid benzyl ester again.In 8.0g is added in a dry 500mL round bottom two-mouth bottle
(0.032mol, 223.23g/mol) aspartic acid benzyl ester, in N2Protection under, the ethyl acetate that newly steams of about 200mL is added,
It flows back at 90 DEG C.The new steaming ethyl acetate solution of 4.2g (0.0142mol, 298.75g/mol) triphosgene is slowly added dropwise into benzyl ester
In solution, at 90 DEG C, it is stirred at reflux until solution becomes clarification.Ice salt bath quenching, quickly with cold saturated sodium bicarbonate solution
Simultaneously sodium chloride solution washed once the saturation of washing three times, cold, extract liquid separation, ethyl acetate layer dry, mistake through anhydrous magnesium sulfate
After filter, concentration, new steaming petroleum ether precipitation is added.After white precipitate filtering, tied again with ethyl acetate/petroleum ether mixed solution
Crystalline substance after solution is cooling, is finally collected and obtains white needle-like crystals.
(2)PEG-PBLA-NH2Synthesis, reaction mechanism and process be as follows:
Weigh 2g PEG-NH2(1mmol) 70 DEG C of vacuum drying 4h are first newly steamed with 20mL after cooling under nitrogen protection
CH2Cl2Dissolution, adds the DMF solution (1.5g is dissolved in 2mL anhydrous DMF) of BLA-NCA, is sufficiently stirred, reaction is begun with
A large amount of minute bubbles generate.Reaction flask is sealed, heating stirring reacts 72h in 35 DEG C of oil bath.After reaction, it is deposited in a large amount of
It in cold anhydrous ether (about 500mL), is washed through centrifugation and anhydrous ether, vacuum drying obtains PEG-PBLA-NH2。
(3) synthesis of PDPA-COOH, reaction mechanism and process are as follows:
The monomer DPA (2.10g, 9.88mmol), chain-transferring agent DDAT of polymerization inhibitor are removed through alkali alumina column
(90.0mg, 0.247mmol) and AIBN (2.0mg, 0.012mmol) are added in the reaction flask of 25mL, and Ar protection is lower to be added
The dioxane dissolution that 10mL newly steams, after all dissolutions, logical Ar gas bell 30min removes the oxygen in system, sealing reaction
Bottle simultaneously reacts 8h at 80 DEG C.After reaction, it is quenched, is then dialysed two days and removed in dioxane with the bag filter of 3500Da
Fall unreacted monomer, then again with water two days removing dioxane of dialysis, finally freeze-drying obtains yellow solid PDPA-DDAT.
PDPA-DDAT 15mL dioxane dissolves, and 20 times of excessive AIBN (0.9g) are added, and until completely dissolved, leads to Ar tympanites
30min deoxygenation is steeped, reacts 8h at 80 DEG C.After reaction, it is quickly cooled to room temperature, then reaction solution is transferred to
The bag filter of 3500Da is dialysed three days removing small molecules in dioxane, is then dialysed two days with water again, and finally freeze-drying obtains
White solid PDPA-COOH.
(4) synthesis of PEG-PAsp (MEA)-PDPA, reaction mechanism and process are as follows:
Triblock polymer is obtained by amidation docking reaction.1.2g PDPA-COOH, 24mg NHS, 40mg DCC are added
Enter the reaction flask to 50mL, and with the CHCl of 20mL3Reaction 1h is stirred at room temperature in dissolution.The PEG-PBLA-NH of 0.71g2With
The CHCl of 10mL3It after dissolution, is added in above-mentioned reaction solution, continues to react two days at room temperature.After reaction, react molten
Liquid is deposited in anhydrous ether after filtering off insoluble matter DCU with syringe filter, collected by suction precipitating, is drained solvent and is obtained white admittedly
Body PEG-PBLA-PDPA.Final product is obtained by PEG-PBLA-PDPA by mercaptoethylmaine ammonolysis reaction.1.5g PEG-PBLA-
PDPA, 0.9g the mercaptoethylmaine DMF of 30mL dissolve, and after reacting 48h at 35 DEG C, reaction solution is with the bag filter of 3500Da in nothing
Dialyse 3d in water methanol, is spin-dried for solvent and obtains white solid final product PEG-PAsp (MEA)-PDPA.
The preparation and characterization of 2 nano-complex of embodiment
The triblock polymer that Example 1 is prepared is dissolved in the PBS of pH 5.0, then presses nano-complex system
Preparation Method prepares nanoparticle, chooses the compound that N/P is 18,24 and 30, measures its hydraulic diameter and surface potential, as a result
As shown in figure 3, triblock polymer partial size does not have apparent pH dependence, respectively less than due to the limitation of subsurface stratum disulfide bond
100nm.The results show that when pH 5.0, acid-sensitive section of tertiary amine of polymer protonates current potential completely, can be compound well by siRNA, multiple
It is positively charged to close object；After pH value becomes 7.4, due to a large amount of tertiary amine deprotonation cause PDPA sections it is hydrophobic, will form similar glue
SiRNA is wrapped in micella core by the structure of beam, at this time nanoparticle surface can because siRNA there are due to be in elecrtonegativity to get arriving
Surface electronegative stable siRNA delivery system；To after the slightly sour microenvironment (pH 6.5) of tumor tissues, connect for PDPA sections at this time
Nearly 50% protonation can have certain compound action with siRNA, and positive electricity is enough, and nanoparticle surface is positively charged.
The result of current potential and partial size confirms the complex systems, in wider N/P than that can obtain in pH 7.4 and 6.5 in range
Under the conditions of have opposite surface electrical behavior nano-complex, be greatly improved tumour enrichment, improve vivo applications efficiency.
SiRNA releasing research in 3 triblock polymer of embodiment
In order to confirm the sensitive release of siRNA, in-vitro simulated release experiment has been carried out.By nano-complex preparation method,
Polymer and FITC-siRNA are prepared into compounds by N/P ratio for 18, and are incubated for respectively in different condition: (1) being same amount
Pure FITC-siRNA；(2)pH 5.0；(3)pH 5.0+10mM GSH；(4)pH 7.4；(5)pH 7.4+5μM GSH；(6)pH
Mixed liquor is cooked into gel blocking electrophoresis experiment after 7.4+10mM GSH, 1.5h.As a result such as Fig. 4, in pH 5.0, even if having highly concentrated
The reducing agent of degree exists, and also the not release (without the band that dissociates) of siRNA, illustrates under lysosomal pH, siRNA is still by polymer
Protection, avoids being degraded by enzymes；Only when pH value rises to 7.4, and when GHS concentration higher (10mM), there is a bright siRNA
Free band is run out of.The experiment be well demonstrated that high reductant concentration and endochylema environment pH be trigger siRNA release two must
Need condition.
The cytotoxicity of 4 triblock polymer of embodiment detects
Triblock polymer is had detected under various concentration to the toxicity of mouse C 6 Cell of Glioma using mtt assay.As a result such as
Shown in Fig. 5, after being incubated for long period (48h), when polymer concentration progressively increases to 500 μ g/mL, do not occur larger proportion yet
Cell death, after polymer concentration reaches 1000 μ g/mL, cell survival rate is substantially reduced, but still has 60% or more to deposit
It is living, illustrate that polymer toxicity is little.MTT experiment is the result shows that polymer itself is little to C6 cytotoxicity, this is for polymerization
Object, which is applied to In vivo study, has apparent advantage compared to traditional cation carrier (PEI, PLL etc.).
5 fluorescent dye with tumour-specific targeting research of embodiment
Using Flow Cytometry carried out quantitative assessment C6 cell to triblock polymer taking the photograph under condition of different pH
Take situation.Experimental result (Fig. 6) display, in pH 7.4, polymer surfaces are negatively charged, repel each other with cell membrane surface negative electricity,
So cell is to it almost without absorption in the short period (8h)；And in pH 6.5, polymer is since surface is positively charged, easily
It is mutually adsorbed with negative electricity albumen on cell membrane, realizes that cell endocytosis, transcriptional efficiency improve nearly 30 times compared to 7.4 groups of pH；Card
Clear tumor tissue environment is conducive to nano-complex by cellular uptake, demonstrates the fluorescent dye with tumour-specific targeting of nano-complex.
Claims (9)
1. a kind of acid-sensitive amphiphilic triblock polymer, which is characterized in that the polymer is by polyethylene glycol section-interlude-acid
Quick section of triblock copolymer is constituted, and the interlude is poly- asparagus fern acyl mercaptoethylmaine, and acid-sensitive section is poly- (diisopropylaminoethyl) first
Base ethyl acrylate, poly- (diisopropylaminoethyl) ethyl methacrylatepKa is 6.3~6.8；Point of the polyethylene glycol section
Son amount is 1000~5000 Da, and the molecular weight of poly- asparagus fern acyl mercaptoethylmaine is 800~2000Da, poly- (diisopropylaminoethyl) first
The molecular weight of base ethyl acrylate is 8000~12000 Da.
2. acid-sensitive amphiphilic triblock polymer according to claim 1, which is characterized in that poly- (the diisopropyl ammonia
Base) ethyl methacrylatepKa is 6.4.
3. acid-sensitive amphiphilic triblock polymer according to claim 1, which is characterized in that point of the polyethylene glycol section
Son amount is 2000 Da, and the molecular weight of poly- asparagus fern acyl mercaptoethylmaine is 1000Da, poly- (diisopropylaminoethyl) ethyl methacrylate
Molecular weight be 10000 Da.
4. the preparation method of the described in any item acid-sensitive amphiphilic triblock polymers of claims 1 to 3, which is characterized in that tool
Body includes the following steps:
S1. using aspartic acid and benzyl alcohol as raw material, using the concentrated sulfuric acid as catalyst, aspartic acid benzyl ester is prepared；
S2. using aspartic acid benzyl ester as raw material, it is slowly added to triphosgene, benzyloxycarbonyl group aspartic acid acid anhydrides is prepared in reflux；
S3. with amino-polyethyleneglycols (PEG-NH2) make macromole evocating agent, cause the acid anhydrides ring-opening polymerisation of benzyloxycarbonyl group aspartic acid
Obtain PEG-PBLA-NH2；
S4. using 2- (dodecyl trithiocarbonic acid ester group) -2 Methylpropionic acid as chain-transferring agent, AIBN makees initiator, causes single
Body 2-(diisopropylaminoethyl) ethyl methacrylate polymerization, reacted after sulphur is sloughed to obtain PDPA- with excess AIBN
COOH；
S5. pass through amidation process for PEG-PBLA-NH2It is docked with PDPA-COOH；
S6. it docks product with cysteamine with above-mentioned final product PEG-PAsp (MEA)-PDPA is obtained by ammonolysis reaction.
5. the described in any item acid-sensitive amphiphilic triblock polymers of claims 1 to 3 are answered in preparation siRNA delivery system
With.
6. the described in any item acid-sensitive amphiphilic triblock polymers of claims 1 to 3 are in preparation cancer diagnosis drug and/or control
Treat the application in drug.
7. a kind of tumour intelligently targets and environment double responsiveness siRNA delivery system, which is characterized in that the siRNA conveys body
System is that siRNA concentration is compound in nanoparticle core by any one of claims 1 to 3 acid-sensitive amphiphilic triblock polymer
It is interior, pH responsiveness is provided by PDPA block tertiary amine, subsurface stratum forms intermolecular disulfide bond by PAsp (MEA) and realizes to siRNA's
It protects and responds siRNA in reproducibility endochylema and discharge.
8. the preparation method of siRNA delivery system described in claim 7, which comprises the steps of: gather three block
It closes object PEG-PAsp (MEA)-PDPA to be dissolved in the buffer solution that pH value is 5.0, and is pressed specifically under the pH value with siRNA
N/P under the conditions of same pH, 30 min of bubble oxygen is passed through into solution and makes the sulfydryl of subsurface stratum full cross-linked than compound 30 min
At disulfide bond, the pH value of solution is finally adjusted to 7.4.
9. intelligently targeting and environment double responsiveness siRNA delivery system are preparing cancer diagnosis drug to tumour described in claim 7
And/or the application in therapeutic agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610559302.1A CN106188555B (en) | 2016-07-12 | 2016-07-12 | A kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system and preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610559302.1A CN106188555B (en) | 2016-07-12 | 2016-07-12 | A kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system and preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106188555A CN106188555A (en) | 2016-12-07 |
| CN106188555B true CN106188555B (en) | 2019-02-05 |
Family
ID=57475932
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610559302.1A Active CN106188555B (en) | 2016-07-12 | 2016-07-12 | A kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system and preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106188555B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106943603B (en) * | 2017-01-24 | 2020-07-14 | 中山大学 | Preparation method of nanogold shell by taking pH sensitive micelle as template |
| CN109897178B (en) * | 2019-02-22 | 2021-06-25 | 临沂大学 | Polyamino acid carboxylic acid water reducing agent and preparation method thereof |
| CN112442179B (en) * | 2019-09-04 | 2021-07-23 | 深圳市第二人民医院 | Amphiphilic polymer for surface biological functionalization and preparation method and application thereof |
| CN110713596B (en) * | 2019-11-08 | 2022-04-29 | 西北师范大学 | PH-reduction dual-response polymer embolic agent for tumor ductless embolization and synthesis thereof |
| CN113694040B (en) * | 2020-05-20 | 2023-05-30 | 中山大学孙逸仙纪念医院 | Therapeutic liposome nanoparticle based on DPPA and preparation method and application thereof |
| CN115885987B (en) * | 2022-09-08 | 2024-06-11 | 河南大学 | Targeting nano-carrier, preparation method and application thereof, targeting drug-loaded nano-carrier and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103755953A (en) * | 2013-12-12 | 2014-04-30 | 深圳先进技术研究院 | Intelligent polycation nano-carrier, and preparation method and application thereof |
| CN103893124A (en) * | 2014-04-24 | 2014-07-02 | 中国药科大学 | Gene-therapy drug delivery system based on cooperative assembling |
| CN103990134A (en) * | 2014-04-22 | 2014-08-20 | 中山大学 | Nano vesicle capable of co-transporting drugs and genes, manufacturing method and applications thereof |
-
2016
- 2016-07-12 CN CN201610559302.1A patent/CN106188555B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103755953A (en) * | 2013-12-12 | 2014-04-30 | 深圳先进技术研究院 | Intelligent polycation nano-carrier, and preparation method and application thereof |
| CN103990134A (en) * | 2014-04-22 | 2014-08-20 | 中山大学 | Nano vesicle capable of co-transporting drugs and genes, manufacturing method and applications thereof |
| CN103893124A (en) * | 2014-04-24 | 2014-07-02 | 中国药科大学 | Gene-therapy drug delivery system based on cooperative assembling |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106188555A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106188555B (en) | A kind of tumour intelligently targeting and environment double responsiveness siRNA delivery system and preparation method and application | |
| Lin et al. | Advances in non-covalent crosslinked polymer micelles for biomedical applications | |
| Li et al. | A composite hydrogel system containing glucose-responsive nanocarriers for oral delivery of insulin | |
| CN104758952B (en) | Nano-carrier of medicine and gene and its production and use is delivered altogether | |
| CN103254442B (en) | Disulfide bond linked polyester comb-type graft copolymer as well as preparation method and application thereof | |
| CN103764153A (en) | Poly(acrylate) polymers for in vivo nucleic acid delivery | |
| CN101787119A (en) | Polymer with tumor organization pH responsiveness and micelle thereof | |
| CN105524272B (en) | The preparation and application of the polyethylene glycol amino acid block copolymer of lipoic acid modification | |
| CN103554508B (en) | Acid-sensitive amphipathic star-block copolymers, its preparation method and application | |
| CN106317416A (en) | Double-pH-response amphiphilic copolymer and preparation method and application thereof | |
| CN105963706B (en) | A kind of branching HPMA copolymer-DOX conjugate and its preparation method and application | |
| CN105524271B (en) | The synthesis and application of the polyamino acid block copolymer of cholic acid modification | |
| CN101480377B (en) | Method for preparing tumor enzyme-targeted nano medicine-carrying polymer microsphere | |
| Chen et al. | Thermal responsive micelles for dual tumor-targeting imaging and therapy | |
| CN110483785A (en) | A kind of triblock polymer, medicament-carried nano micelle, Nano medication and its preparation method and application | |
| CN103251596A (en) | 7-ethyl-10-hydroxycamptothecin amphiphilic polymer prodrug as well as preparation method and nano-particles thereof | |
| CN107641181A (en) | A kind of di-block copolymer with light and pH dual responsiveness and preparation method thereof | |
| CN106995516A (en) | Nanometer medicine-carried system of tumour-specific enrichment and preparation method thereof | |
| CN104725581A (en) | Method for preparing and applying light/temperature sensitive amphiphilic block polymer micelle | |
| CN105327362B (en) | A kind of preparation method of the graphene targetable drug carriers of amphipathic nature polyalcohol brush modification | |
| CN105085845A (en) | Polymer, preparation method and poly-N-isopropylacrylamide europium complex micelle | |
| CN105663033B (en) | Intratumor injection temperature sensitive polymer gel nano-composition in situ and application | |
| CN107501547A (en) | A kind of phenyl boric acid functionalization amphion block copolymer and glucose-sensitive bionic nano carrier | |
| CN104027816B (en) | Co-loaded adriamycin and siRNA (small interfering ribose nucleic acid) carrier capable of removing pegylation and synthesis method thereof | |
| CN108395543A (en) | A kind of modified polyrotaxane, the carrier micelle and the preparation method and application thereof based on polyrotaxane |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |