CN108219089B - Target the preparation of multifunctional polymer nano-probe and its to the application in cell imaging containing TK - Google Patents
Target the preparation of multifunctional polymer nano-probe and its to the application in cell imaging containing TK Download PDFInfo
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- CN108219089B CN108219089B CN201810059203.6A CN201810059203A CN108219089B CN 108219089 B CN108219089 B CN 108219089B CN 201810059203 A CN201810059203 A CN 201810059203A CN 108219089 B CN108219089 B CN 108219089B
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- 229920000642 polymer Polymers 0.000 title claims abstract description 51
- 238000003384 imaging method Methods 0.000 title claims abstract description 19
- 239000000523 sample Substances 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 239000002105 nanoparticle Substances 0.000 claims abstract description 18
- JNTOCHDNEULJHD-UHFFFAOYSA-N Penciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(CCC(CO)CO)C=N2 JNTOCHDNEULJHD-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229960001179 penciclovir Drugs 0.000 claims abstract description 14
- -1 mercapto alkene Chemical class 0.000 claims abstract description 12
- 238000012650 click reaction Methods 0.000 claims abstract description 6
- 229940079593 drug Drugs 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims abstract description 5
- 108700008625 Reporter Genes Proteins 0.000 claims abstract description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 7
- 125000004185 ester group Chemical group 0.000 claims description 6
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims description 4
- 229960003823 gadoteric acid Drugs 0.000 claims description 4
- 238000002595 magnetic resonance imaging Methods 0.000 claims description 4
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 229910021645 metal ion Inorganic materials 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- IZUPBVBPLAPZRR-UHFFFAOYSA-N pentachloro-phenol Natural products OC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl IZUPBVBPLAPZRR-UHFFFAOYSA-N 0.000 claims description 3
- 150000007970 thio esters Chemical class 0.000 claims description 3
- 238000003325 tomography Methods 0.000 claims description 3
- RFOWDPMCXHVGET-UHFFFAOYSA-N (2,3,4,5,6-pentafluorophenyl) prop-2-enoate Chemical compound FC1=C(F)C(F)=C(OC(=O)C=C)C(F)=C1F RFOWDPMCXHVGET-UHFFFAOYSA-N 0.000 claims description 2
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical group N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims description 2
- 229930012538 Paclitaxel Natural products 0.000 claims description 2
- 229940009456 adriamycin Drugs 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000007334 copolymerization reaction Methods 0.000 claims description 2
- 238000004043 dyeing Methods 0.000 claims description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 2
- 238000000799 fluorescence microscopy Methods 0.000 claims description 2
- GFSTXYOTEVLASN-UHFFFAOYSA-K gadoteric acid Chemical compound [Gd+3].OC(=O)CN1CCN(CC([O-])=O)CCN(CC([O-])=O)CCN(CC([O-])=O)CC1 GFSTXYOTEVLASN-UHFFFAOYSA-K 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 238000006384 oligomerization reaction Methods 0.000 claims description 2
- 229960001592 paclitaxel Drugs 0.000 claims description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- QEWYKACRFQMRMB-UHFFFAOYSA-N fluoroacetic acid Chemical compound OC(=O)CF QEWYKACRFQMRMB-UHFFFAOYSA-N 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- 238000002242 deionisation method Methods 0.000 claims 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims 1
- 239000010931 gold Substances 0.000 claims 1
- 229910052737 gold Inorganic materials 0.000 claims 1
- 150000002500 ions Chemical class 0.000 claims 1
- 238000005915 ammonolysis reaction Methods 0.000 abstract description 6
- 239000007864 aqueous solution Substances 0.000 abstract description 4
- 230000008685 targeting Effects 0.000 abstract description 4
- 238000012712 reversible addition−fragmentation chain-transfer polymerization Methods 0.000 abstract description 3
- 238000007306 functionalization reaction Methods 0.000 abstract description 2
- 238000011938 amidation process Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 22
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 239000003208 petroleum Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 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 description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
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- 238000005516 engineering process Methods 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000001338 self-assembly Methods 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 229910003317 GdCl3 Inorganic materials 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 2
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- 230000000694 effects Effects 0.000 description 2
- MEANOSLIBWSCIT-UHFFFAOYSA-K gadolinium trichloride Chemical compound Cl[Gd](Cl)Cl MEANOSLIBWSCIT-UHFFFAOYSA-K 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- YPJUNDFVDDCYIH-UHFFFAOYSA-N perfluorobutyric acid Chemical compound OC(=O)C(F)(F)C(F)(F)C(F)(F)F YPJUNDFVDDCYIH-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- XBNGYFFABRKICK-UHFFFAOYSA-N 2,3,4,5,6-pentafluorophenol Chemical compound OC1=C(F)C(F)=C(F)C(F)=C1F XBNGYFFABRKICK-UHFFFAOYSA-N 0.000 description 1
- UGIJCMNGQCUTPI-UHFFFAOYSA-N 2-aminoethyl prop-2-enoate Chemical compound NCCOC(=O)C=C UGIJCMNGQCUTPI-UHFFFAOYSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 238000012307 MRI technique Methods 0.000 description 1
- DXRFZHILMCWCNG-UHFFFAOYSA-N N,N-dimethyl-1,8-naphthyridin-2-amine Chemical compound C1=CC=NC2=NC(N(C)C)=CC=C21 DXRFZHILMCWCNG-UHFFFAOYSA-N 0.000 description 1
- 108010011356 Nucleoside phosphotransferase Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012879 PET imaging Methods 0.000 description 1
- 101150003725 TK gene Proteins 0.000 description 1
- HFBMWMNUJJDEQZ-UHFFFAOYSA-N acryloyl chloride Chemical compound ClC(=O)C=C HFBMWMNUJJDEQZ-UHFFFAOYSA-N 0.000 description 1
- 239000001361 adipic acid Substances 0.000 description 1
- 235000011037 adipic acid Nutrition 0.000 description 1
- FTWHFXMUJQRNBK-UHFFFAOYSA-N alpha-Methylen-gamma-aminobuttersaeure Natural products NCCC(=C)C(O)=O FTWHFXMUJQRNBK-UHFFFAOYSA-N 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000012711 chain transfer polymerization Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
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- 229910021641 deionized water Inorganic materials 0.000 description 1
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- 239000012046 mixed solvent Substances 0.000 description 1
- 238000003333 near-infrared imaging Methods 0.000 description 1
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- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
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- 206010033675 panniculitis Diseases 0.000 description 1
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
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- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
- A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
- A61K49/106—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being cyclic, e.g. DOTA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
- A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
-
- 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
- C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
- C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
-
- 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
- C08F8/00—Chemical modification by after-treatment
- C08F8/44—Preparation of metal salts or ammonium salts
Abstract
The invention discloses a kind of preparation of targeting multifunctional polymer nano-probe and its to the application in cell imaging containing TK.The present invention obtains a kind of novel amphiphilic block polymer by the way of RAFT polymerization, modifies after carrying out functionalization to polymer by ammonolysis and amidation process.Using mercapto alkene click-reaction, Penciclovir drug can be keyed to polymer ends, obtain target polymer, target polymer can carry out specific recognition to the cell of the reporter gene containing TK after being self-assembled into nanoparticle in aqueous solution.
Description
Technical field
The invention belongs to high molecular material and bio-imaging field, it is related to a kind of novel amphiphilic block polymer, thereafter
Modified outcome and application, especially a kind of novel targeted multifunctional polymer nanoparticle and its preparation transfect carefully with it to TK
Application in the imaging of born of the same parents' specificity.
Background technique
Currently used i mage analysis methods have magnetic resonance imaging (MRI), optical imagery, radionuclide image.However,
In molecular image technologies all at present, a variety of different image technologies are respectively provided with respective advantage, but are different aobvious
The shortcomings that overcoming or limitation itself are difficult to as technology also exists simultaneously.Optical imagery is capable of providing on cell and molecular level high
The structural information of the resolution ratio penetrability limitation that still optics images, can only be imaged subcutaneous tissue；MRI technique is to this
Not limited, it can provide the structural information of 3 D anatomical formula, but remolding sensitivity is lower.Radionuclide image is sensitive
Degree is very high, but resolution ratio is poor.Therefore, two kinds or more of imaging modes are merged and carries out bimodal or multi-modality imaging, it is right
Develop highly sensitive, high specific tracing method to have great importance.
So far, the nanoparticle of polymer material is widely used in biological field, this is because polymer is with following
Advantage: (1) polymer molecular weight is big, drug can be made in lesions position stay longer as carrier；It (2) can be some tools
There are targeting or treatment imaging component to be integrated on polymer by way of chemical bonding；(3) polymer and catabolite
It is small to body toxic side effect, it is avoided that carrier material gathers in human organ tissue, generates toxic side effect.Amphipathic block is total
Polymers can be self-assembly of micella and a kind of polymer nano-particle in aqueous solution.The distribution of these Nanoparticle Sizes
It is very narrow, there is core-shell structure, aggregation forms the kernel of particle between hydrophobic segment, and shell then forms brush knot by hydrophilic segment
Structure, these hydrophilic segments usually have biocompatibility and have been dispersed in water three-dimensional stabilization to particle, in hydrophilic segment
End can also introduce the component with target function.
Herpes simplex thymidine nucleoside kinase (HSV1-TK) gene is report relatively conventional in rii gene technology
Accuse gene.Nucleoside analog phosphoric acid can be turned to 5- phosphoric acid nucleoside by it, and 5- phosphoric acid nucleoside is because not across cell membrane, so will
It is trapped in cell, realizes the aggregation in specific TK transfection cell.18The Penciclovir small-molecule drug that F replaces is in positive electron
Emission computed tomography (PET) tracer TK is transfected and is achieved very high contrast in cell, but grinding as targeting group
Study carefully and does not report but.
The invention discloses a kind of novel targeted multifunctional polymer nanoparticles and preparation method thereof.Using it is reversible-plus
A kind of novel amphiphilic block polymer is obtained at the mode of-fracture-chain transfer polymerization (RAFT polymerization), passes through ammonolysis and amide
Change after reaction carries out functionalization to polymer and modifies.Using mercapto alkene click-reaction, Penciclovir drug is keyed to polymer end
End.Specific recognition can be carried out to the cell of the reporter gene containing TK after being self-assembled into nanoparticle in aqueous solution.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of novel amphiphilic block polymer, thereafter
Modified outcome and its application especially target multifunctional polymer nano-probe and its in TK transfection cell-specific imaging
Application.
Novel amphiphilic block polymer is that the chain-transferring agent containing three thioesters is used to obtain first in a manner of RAFT polymerization
Block be gather boc-protected 2- aminoacrylic acid ethyl ester (BEA), the second block be oligomerization PEG acrylate monomer (OEGA) and
The block polymer of Pentafluorophenol acrylate random copolymerization；The structural formula of the novel amphiphilic block polymer are as follows:
Wherein, R1ForR2Form、n、x、y
It is the degree of polymerization, can use arbitrary value.
It is modified after obtained block polymer is carried out: by the amino-containing function of the Pentafluorophenol ester group in polymer
Molecule carries out ammonolysis formation amide and obtains product 1 so that functional molecular is keyed on polymer lateral chain；It again will be in product 1
BEA segment carries out de- boc protection in trifluoroacetic acid, and obtained amino is reacted with the molecule containing ester group or aldehyde radical, obtains product
2.Preferably, the amino-containing functional molecular is amino acid, amino Gadoteric Acid, adriamycin or taxol.Preferably, institute
The molecule containing ester group or aldehyde radical stated is rhodamine, fluorescein, bipyridyl or camptothecine.
For being modified after above-mentioned polymer, when the functional molecular uses Gadoteric Acid DOTA, i.e., with amino-DOTA ammonolysis
Phenyl-pentafluoride phenolic ester obtains the polymer of the i.e. DOTA of product 1 modification, can be with metal ion (Gd3+,68Ga3+, or111In3+Deng) into
Row coordination is used for magnetic resonance imaging, positron emission computerized tomography or single photon emission computerized tomography,SPECT as probe
(SPECT)；When amino progress amidation of the molecule containing ester group or aldehyde radical using cyanine dye Cy5.5, with hydrolysis
Reaction can be used as probe for near-infrared fluorescence imaging.
For pentafluorophenyl esters by during ammonolysis, three thioesters of end group also can be sulfydryl by ammonolysis, sulfydryl can with contain double bond
Compound carry out mercapto alkene click-reaction.
By above-mentioned mercapto alkene click-reaction, Penciclovir drug can be keyed to polymer ends, it is poly- to obtain targeting
Object is closed, disperses target polymer in deionized water and is assembled into nanoparticle, is used to report base to containing TK as nano-probe
The cell of cause carries out specific imaging.
The beneficial effects of the present invention are:
The present invention devises a kind of novel Amphipathilic block polymer, and modifies after carrying out to it, in aqueous solution may be used
It is self-assembly of the nanoparticle of partial size 30nm or so.The product modified after carrying out to above-mentioned block polymer can be applied to closely
In infrared imaging, magnetic resonance imaging and PET imaging.In addition, obtaining target polymer by introducing Penciclovir, it may make and receive
Rice corpuscles has specific recognition to the cell that TK is transfected, to carry out the multi-modal imaging of specificity.
Detailed description of the invention
Fig. 1 is the synthetic route of PBEA.
Fig. 2 is the synthetic route of PBEA-b-POEGAPF.
Fig. 3 is the GPC curve of PBEA and PBEA-b-POEGAPF.
Fig. 4 is the schematic diagram of modification and self assembly after PBEA-b-POEGAPF.
Fig. 5 is the grain size distribution of the nanoparticle of self assembly.
Fig. 6 is laser co-focusing image of the nanoparticle to two kinds of 293T cell imagings.
Fig. 7 is the laser co-focusing image that nanoparticle transfects 293T cell imaging to TK.
Specific embodiment
Instrument and material
2- aminoethyl acrylate, di-tert-butyl dicarbonate, OEGA, Pentafluorophenol, acryloyl chloride, dimethylamino naphthyridine
(DMAP), azodiisobutyronitrile (AIBN), Penciclovir (PCV), GdCl3·6H2O etc. is purchased from lark prestige Reagent Company.Dimethyl
Formamide (DMF), petroleum ether, anhydrous ether, methylene chloride, triethylamine etc. are purchased from traditional Chinese medicines chemical reagent Co., Ltd.
Embodiment 1
The synthesis step of block polymer:
1) synthesis of PBEA
215mg (1mmol) BEA 1.1mg (0.006mmol) AIBN, 9.0mg (0.03mmol) CTA is weighed to be dissolved in
In the THF of 1.0mL.Polymerization bottle is freezed-is vacuumized-thaw cycles three times, then pass to nitrogen, be put into 65 DEG C of oil baths and react
8h.Reaction is terminated in liquid nitrogen afterwards, is diluted with THF, is then precipitated in petroleum ether, is centrifugated.So dissolution precipitates three times,
Obtain colorless viscous polymer 135mg, drying at room temperature under vacuum condition.2) synthesis of PBEA-b-PFOEG
58mg PBEA, 240mg (0.5mmol) OEGA, 24mg (0.1mmol) PFBA, 0.5mg (0.003mmol) AIBN is molten
Solution is in the THF of 1.0mL, PBEA:OEGA:PFBA=1:50:10.Polymerization bottle is freezed-is vacuumized-thaw cycles three times, so
After be passed through nitrogen, be put into 65 DEG C of oil baths and react 8h.Reaction is terminated in liquid nitrogen afterwards, is diluted with THF, is then sunk in petroleum ether
It forms sediment, centrifuge separation.So dissolution precipitating three times, obtains colorless viscous polymer 240mg, drying at room temperature under vacuum condition.
Embodiment 2
The rear modification step of block polymer:
1) the rear modification of DOTA
159mg PBEA-b-PFOEG is weighed, 39mg amino-DOTA, 80mg triethylamine is dissolved in methylene chloride, and room temperature is anti-
It should be precipitated in petroleum ether for 24 hours, centrifugation obtains the polymer of DOTA modification, which is verified by nuclear-magnetism.
2) the rear modification of Cy5.5 and PCV
Step 1) resulting polymers are dissolved in the in the mixed solvent of 1ml trifluoroacetic acid and methylene chloride, react at room temperature 4h, it will
Solvent revolving removes, and add methylene chloride dissolution, precipitates in ether, and centrifugation obtains taking off boc-protected polymer.Then by it
In methylene chloride, 7mg Cy5.5, the double NHS esters of 17mg adipic acid, the Penciclovir of 12.5mg acrylate modification is added in dissolution
With 36.6mg DMAP, while the modification of fluorescent dye is carried out, the crosslinking of polymer and the mercapto alkene click-reaction of PCV.Reaction is for 24 hours
It is precipitated in petroleum ether afterwards, obtains nanoparticle.
3) the rear modification of metal ion
By the above-mentioned nanoparticle of 100mg and 326mg GdCl3·6H2O is dissolved in 1mL DMF, is stirred at room temperature for 24 hours.Then slowly
5ml water is added, dialysis removes extra DMF, the nano-particle solution dispersed in water.
Embodiment 3
Cell imaging experiment
The virus of the gene containing TK is added in 293T cell, so that cell is contained TK reporter gene using virus infection, then removes
Culture medium and virus are removed, 495ul new culture medium and 5ul nano-particle solution is added, co-cultivation removes culture medium afterwards for 24 hours, uses
Culture medium washing, carries out dyeing patch, carries out laser co-focusing test.Image results are as shown in fig. 6, because the cell of TK transfection
In contain green fluorescent protein (GFP), so the picture left above has green fluorescence, and right side normal cell does not have then, it was demonstrated that TK gene
Successful transfection.By two figures of comparison left and right, the cell imaging that the nano-probe of PCV can transfect TK is modified, and it is normal thin
Born of the same parents are then without fluorescence signal.Illustrate that the nanoparticle for modifying PCV is easy to be detained in the cell that TK is transfected, TK is turned to realize
Contaminate the specificity imaging of cell.Fig. 7 is the 293T cell in same panel region containing transfection and untransfected, by comparing us
It was found that the cell containing GFP imaging can equally carry out the near-infrared imaging of Cy5.5, and there is not can be carried out then for GFP close red
Outer imaging, it was demonstrated that the specific binding of nano-probe containing PCV and tk transfection cell.
Claims (10)
1. a kind of Amphipathilic block polymer, which is characterized in that be the side for using the chain-transferring agent containing three thioesters to polymerize with RAFT
Formula obtain the first block be gather boc-protected acrylic acid -2- amino ethyl ester BEA, the second block is oligomerization PEG acrylate monomer
The block polymer of OEGA and the copolymerization of Pentafluorophenol acrylate random；The structural formula of the Amphipathilic block polymer are as follows:
。
2. a kind of rear modified outcome based on Amphipathilic block polymer as described in claim 1, which is characterized in that through as follows
Method obtains: the Pentafluorophenol ester group in polymer as described in claim 1 is carried out ammonia with amino-containing functional molecular
Solution forms amide, so that functional molecular is keyed on polymer lateral chain, obtains product 1；Again by BEA segment in product 1 three
De- boc protection is carried out in fluoroacetic acid, obtained amino is reacted with the molecule containing ester group or aldehyde radical, obtains product 2.
3. modified outcome after according to claim 2, which is characterized in that the amino-containing functional molecular is amino
Acid, amino Gadoteric Acid, adriamycin or taxol.
4. modified outcome after according to claim 2, which is characterized in that the functional molecular uses Gadoteric Acid DOTA.
5. a kind of application of rear modified outcome as claimed in claim 4, which is characterized in that by the corresponding product 1 of acquisition and gold
Belong to disconnected for magnetic resonance imaging, positron emission computerized tomography or single photon emission computed as probe after ion is coordinated
Layer scanning, the metal ion are Gd3+、68Ga3+Or111In3+。
6. modified outcome after according to claim 2, which is characterized in that the molecule containing ester group or aldehyde radical is Luo Dan
Bright, fluorescein, bipyridyl or camptothecine.
7. modified outcome after according to claim 2, which is characterized in that the molecule containing ester group or aldehyde radical is cyanine
Uniformly dyeing material Cy5.5.
8. a kind of application of rear modified outcome as claimed in claim 7, which is characterized in that regard corresponding product 2 as probe
For near-infrared fluorescence imaging.
9. a kind of application of rear modified outcome as claimed in claim 7, which is characterized in that by corresponding product 2 and contain double bond
Penciclovir drug carry out mercapto alkene click-reaction, obtain target polymer.
10. the application of modified outcome after as claimed in claim 9, which is characterized in that disperse deionization for target polymer
It is assembled into nanoparticle in water, is used to carry out specific imaging to the cell of the reporter gene containing TK as nano-probe.
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