CN107158410A - A kind of folic acid chitosan Cy7 polymer with tumor-targeting and preparation method thereof - Google Patents
A kind of folic acid chitosan Cy7 polymer with tumor-targeting and preparation method thereof Download PDFInfo
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- CN107158410A CN107158410A CN201710111439.5A CN201710111439A CN107158410A CN 107158410 A CN107158410 A CN 107158410A CN 201710111439 A CN201710111439 A CN 201710111439A CN 107158410 A CN107158410 A CN 107158410A
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- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 53
- 229920000642 polymer Polymers 0.000 title claims abstract description 42
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 36
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 33
- 239000011724 folic acid Substances 0.000 title claims abstract description 33
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 229960000304 folic acid Drugs 0.000 title claims abstract description 20
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 46
- 239000002105 nanoparticle Substances 0.000 claims abstract description 20
- 238000001338 self-assembly Methods 0.000 claims abstract description 10
- GDIYMWAMJKRXRE-UHFFFAOYSA-N (2z)-2-[(2e)-2-[2-chloro-3-[(z)-2-(1,3,3-trimethylindol-1-ium-2-yl)ethenyl]cyclohex-2-en-1-ylidene]ethylidene]-1,3,3-trimethylindole Chemical class CC1(C)C2=CC=CC=C2N(C)C1=CC=C1C(Cl)=C(C=CC=2C(C3=CC=CC=C3[N+]=2C)(C)C)CCC1 GDIYMWAMJKRXRE-UHFFFAOYSA-N 0.000 claims abstract description 4
- 150000002224 folic acids Chemical class 0.000 claims abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 55
- 239000000047 product Substances 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 8
- 125000001246 bromo group Chemical group Br* 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- LGRFSURHDFAFJT-UHFFFAOYSA-N Phthalic anhydride Natural products C1=CC=C2C(=O)OC(=O)C2=C1 LGRFSURHDFAFJT-UHFFFAOYSA-N 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 239000008187 granular material Substances 0.000 claims description 4
- SYBYTAAJFKOIEJ-UHFFFAOYSA-N 3-Methylbutan-2-one Chemical compound CC(C)C(C)=O SYBYTAAJFKOIEJ-UHFFFAOYSA-N 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 claims description 3
- 229940067157 phenylhydrazine Drugs 0.000 claims description 3
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 3
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims description 2
- 238000012650 click reaction Methods 0.000 claims description 2
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 claims description 2
- JKANAVGODYYCQF-UHFFFAOYSA-N prop-2-yn-1-amine Chemical compound NCC#C JKANAVGODYYCQF-UHFFFAOYSA-N 0.000 claims description 2
- 239000006227 byproduct Substances 0.000 claims 1
- 210000004881 tumor cell Anatomy 0.000 abstract description 12
- 229940064302 folacin Drugs 0.000 abstract description 11
- 230000003287 optical effect Effects 0.000 abstract description 10
- 238000002560 therapeutic procedure Methods 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 8
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 8
- 239000003795 chemical substances by application Substances 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 238000011282 treatment Methods 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 229940014144 folate Drugs 0.000 abstract description 2
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical compound [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 39
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 238000010521 absorption reaction Methods 0.000 description 13
- 229910052757 nitrogen Inorganic materials 0.000 description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 239000007787 solid Substances 0.000 description 11
- 125000000304 alkynyl group Chemical group 0.000 description 9
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000003384 imaging method Methods 0.000 description 7
- 230000001988 toxicity Effects 0.000 description 7
- 231100000419 toxicity Toxicity 0.000 description 7
- 150000003852 triazoles Chemical group 0.000 description 7
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 6
- 238000002329 infrared spectrum Methods 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 5
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000002539 nanocarrier Substances 0.000 description 3
- 238000002428 photodynamic therapy Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- 235000019187 sodium-L-ascorbate Nutrition 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000007900 aqueous suspension Substances 0.000 description 2
- JHIWVOJDXOSYLW-UHFFFAOYSA-N butyl 2,2-difluorocyclopropane-1-carboxylate Chemical compound CCCCOC(=O)C1CC1(F)F JHIWVOJDXOSYLW-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- 230000006196 deacetylation Effects 0.000 description 2
- 238000003381 deacetylation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001235 sensitizing effect Effects 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 206010013457 Dissociation Diseases 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 208000018459 dissociative disease Diseases 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000002603 single-photon emission computed tomography Methods 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- 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
-
- 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
- A61K49/0052—Small organic molecules
Abstract
The invention discloses a kind of new folic acid chitosan Cy7 polymer as diagnosis and treatment agent(CF7)And preparation method thereof.The derivative is by the deoxidation N phthalimide-baseds chitosan of 6 nitrine 6 and alkynyl-modified folic acid(ALK‑FA)With alkynyl-modified heptamethine cyanine Cy7(ALK‑Cy7)Reaction synthesis.The polymer CF7 of the present invention can be self-assembly of nanoparticle, can be used as the folate molecule that is coupled on the nanometer diagnosis and treatment agent of tumour, its skeleton can the height expression of targets identification folacin receptor tumour cell, Cy7 molecules can be used for near-infrared fluorescence imaging and optical dynamic therapy.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to a kind of folic acid-chitosan-Cy7 polymer(CF7)And its prepare
Method and purposes, further relate to nanoparticle for being formed by the polymer and its production and use.
Background technology
Cancer is the one of the main reasons of current threat human health.Traditional chemotherapy can give normal cell and tissue
Infringement is brought, the maximum difficulty as oncotherapy.Due between tumor tissues and normal structure in terms of pathology and physiological characteristic
Significant difference is there is, tumor locus vascular permeability enhancing, macromolecular drug, nano-carrier etc. readily penetrates through intravascular
Chrotoplast enters tumor tissues, and due to removing obstacles, can for a long time, high concentration be accumulated in tumor locus, this effect is referred to as
Infiltration and delay enhancing(EPR)Effect(Ghaz-Jahanian, M. A.; Abbaspour-Aghdam, F.; Anarjan,
N.; Berenjian, A.; Jafarizadeh-Malmiri, H., Application of Chitosan-Based
Nanocarriers in Tumor-Targeted Drug Delivery. Molecular Biotechnology 2015,
57, (3), 201-218.).There are a variety of specific receptors, such as folacin receptor on still further aspect, tumor tissue cell's film.
The targeted therapy of tumour cell be by the use of tumor cell surface specific antigen and acceptor as target spot treatment method.Big portion
Divide tumor cell surface height expression folacin receptor(FR), and normal cell surface low expression folacin receptor.We can utilize leaf
Acid coupling cancer therapy drug is so as to reach the purpose of active targeting tumour cell.
Near-infrared(NIR)The fluorescent dye tissue permeability good due to being provided with, the near infrared light of absorption is at biological group
Penetration depth in knitting is larger, and the fluorescence excited is influenceed smaller in itself by biological tissue, so detectable deep tissues
Fluorescence signal.Such dyestuff has good application as the molecular image reagent of Noninvasive in the early detection of cancer
Prospect.Wherein most representational is near-infrared cyanine dye.Heptamethine cyanine(Heptamethine cyanine, Cy7)
It is the fluorescent marker dyes of one of which function admirable, molar absorption coefficient is highest in fluorescent dye, is widely used in
Albumen, antibody, mark and the detection of nucleic acid and other biological molecule(Xiao, L.; Zhang, Y.; Berr, S. S.;
Chordia, M. D.; Pramoonjago, P.; Pu, L.; Pan, D., A novel near-infrared
fluorescence imaging probe for in vivo neutrophil tracking. Molecular imaging
2012, 11, (5), 372-82.).Photodynamic therapy(Photodynamic Therapy, PDT)It is to utilize photodynamic effect
Carry out a kind of new technology of medical diagnosis on disease and treatment.Its exposure basis is photodynamic effect.Its process is, the laser of specific wavelength
Irradiation is excited the sensitising agent of tissue resorption, and oxygen of the sensitising agent of excitation state energy transmission to surrounding, and generation is lived
Property very strong singlet oxygen, singlet oxygen and adjacent large biological molecule occur oxidation reaction, produce cytotoxic effect, and then cause
Cell damage or even death.Cy7 is used for optical dynamic therapy by many researchs existing at present.So we select
Cy7 is used as near-infrared fluorescence imaging and optical dynamic therapy.
Chitosan(Chemical name:β-(1 → 4) -2-amino-2-deoxy-D-Glucose)Be by nature be widely present it is several
Ding Zhi(chitin)Obtained by deacetylation.Chitosan has good biocompatibility, biological degradability and low toxicity
The features such as property.Existing amino has hydroxyl again in its structure, it is easy to chemical modification, is a kind of novel medicine with wide application prospect
Thing carrier.In addition chitosan is also widely used for biomedicine field, such as organizational project, wound healing, bio-imaging and medicine
Conveying etc..But it is due to chitosan poor solubility, so to be chemically modified to it.Therefore current most of documents are at it
2 upper introducing functional groups, increase its solubility.
Nobel chemistry Prize winner Sharpless in 2001 proposes " Click " chemical reaction.It represents reaction as by end
Monomer reaction formation compound with triazole ring structure of the end with alkynyl and azido group.The reaction is quick, efficient and has
There is high selectivity, be widely used in synthesizing various materials.
Based on background above, the present invention devises a kind of polymer, the polymer by Azide chitosan derivatives with
The folic acid of alkynyl(ALK-FA)And the Cy7 of alkynyl(ALK-Cy7)Chemically reacted and be coupled by Click, and can be by certainly
Assembling forms nanoparticle.By connecting FA and Cy7, make it that not only there is the ability for efficiently recognizing target tumour cell, and can use
In near-infrared fluorescence imaging and optical dynamic therapy.
The content of the invention
Based on above research background, the present inventor utilizes " Click " reaction by ALK-FA and ALK-Cy7 and 6- nitrine -6-
'-deoxy-n-phthalimide-based-chitosan reaction, synthesizes folic acid-chitosan-Cy7 polymer(CF7).The present invention's is poly-
Compound CF7 can be self-assembly of nanoparticle, and the folate molecule that can be used as being coupled on the nanometer diagnosis and treatment agent of tumour, its skeleton can be targetted
The tumour cell of folacin receptor height expression is recognized, Cy7 molecules can be used for near-infrared fluorescence imaging and optical dynamic therapy.
Therefore the invention aims to provide a kind of polymer CF7 and preparation method thereof.Another object of the present invention
It is to provide nanoparticle of polymer formation and its production and use.
The invention provides a kind of polymer CF7, its structural formula is:
Wherein n is the number of chitosan derivatives repeat unit.
The polymer CF7 of the present invention can be prepared by the following method, and reaction equation is as follows:
Wherein n is the number of chitosan derivatives repeat unit.
In reaction equation, 1 is chitosan;2 be N- O-phthalic imido grpup chitosans;3 be the bromo- 6- '-deoxy-ns-neighbour's benzene two of 6-
Carboximide base-chitosan;4 be 6- nitrine -6- '-deoxy-ns-phthalimide-based-chitosan;5 be polymer CF7.
The CF7 of present invention synthetic method, comprises the following steps:
Step a:Chitosan 1 is weighed, with phthalic anhydride substituted-amino, N- O-phthalic imido grpups chitosan 2 is obtained;
Step b:6 hydroxyls on product 2 are subjected to bromine substitution reaction, the bromo- 6- '-deoxy-ns of product 6--phthalyl are obtained sub-
Amido-chitosan 3;
Step c:6 bromines on product 3 are subjected to azido substitution reaction, 6- nitrine -6- '-deoxy-ns-phthalyl is obtained
Imido grpup-chitosan 4;
Step d:Product 4 and ALK-FA and ALK-Cy7 are carried out under the catalytic action of anhydrous cupric sulfate and vitamin C sodium salt
Click reacts, and obtains product 5;Wherein ALK-FA is obtained by folic acid and propargylamine reaction(Guo, Z.; Zhang, P.;
Song, M.; Wu, X.; Liu, C.; Zhao, Z.; Lu, J.; Zhang, X., Synthesis and
preliminary evaluation of novel Tc-99m-labeled folate derivative via click
reaction for SPECT imaging. Applied Radiation And Isotopes 2014, 91, 24-30),
ALK-Cy7 is to be obtained by phenylhydrazine and 3- methyl -2- butanone by series reaction(Yang, Z.; Lee, J. H.; Jeon,
H. M.; Han, J. H.; Park, N.; He, Y.; Lee, H.; Hong, K. S.; Kang, C.; Kim, J.
S., Folate-Based Near-Infrared Fluorescent Theranostic Gemcitabine Delivery.J Am Chem Soc 2013, 135, (31), 11657-11662).
The chitosan 1 used in the present invention(Cs)Weight average molecular weight be 10-1000 kilodaltons.
The specific reactions steps of polymer CF7 of the present invention are as follows:
Step a:Chitosan 1 is weighed, dry DMF is dissolved in, phthalic anhydride is added, nitrogen protection, 120 DEG C of oil bath stirrings add
Heat.At the end of reaction, reaction solution is poured into frozen water, yellow-white precipitation is separated out.Suction filtration, solid is washed with ether, acetone, is done
It is dry, obtain N- O-phthalic imido grpups chitosan 2;
Step b:Product 2 is weighed, 1-METHYLPYRROLIDONE is dissolved in(NMP), add N- bromo-succinimides(NBS)And triphen
Base phosphine(TPP).Under nitrogen protection, 80 DEG C of two hours of reaction.After reaction terminates, reaction solution is poured into ethanol, separated out solid
Body.Centrifugation, collects product, and is respectively cleaned three times and dried with ethanol, acetone, obtains brown-red solid 3;
Step c:3 are weighed, 1-METHYLPYRROLIDONE is dissolved in.Add sodium azide(NaN3), nitrogen protection, 80 DEG C of reactions 4
Hour.After reaction terminates, reaction solution is poured into ethanol, solid is separated out.Centrifugation, collects product, product is successively with ethanol, secondary
Water, acetone are respectively washed three times.It is dried to obtain brown solid 4;
Step d:Product 4 is dissolved in dimethyl sulfoxide (DMSO)(DMSO), ALK-FA and ALK-Cy7 is then added, nitrogen is protected, then
Anhydrous cupric sulfate and vitamin C sodium salt are dissolved in water, beaker is slowly added dropwise to.80 DEG C of 72 hours of reaction., will after reaction terminates
Reaction solution is added in bag filter, with pure water dialysis 72h, is freezed, is obtained product 5(CF7);Wherein ALK-FA is by folic acid and third
Ynamine reaction is obtained, and ALK-Cy7 is to be obtained by phenylhydrazine and 3- methyl -2- butanone by series reaction.
Heretofore described polymer CF7 molecular weight is 100-1000 kilodaltons.
In step d, product 4 and ALK-FA and ALK-Cy7 mass ratio are:2∶1∶1.Bag filter molecular cut off is
10000~14000。
The heretofore described polymer can form nanoparticle and preparation method thereof.This method is by polymer CF7
It is dissolved in dimethyl sulfoxide (DMSO), then it is slowly added dropwise in the beaker equipped with pure water with syringe, stirring mixing, room temperature is quiet
Put.The polymer is by being self-assembly of nanoparticle.Concretely comprise the following steps:By heretofore described polymer dimethyl sulfoxide (DMSO)
0.1 ~ 1 mg/ml solution is made into, then 1 milliliter is drawn with syringe, it is slowly added dropwise to equipped with 20 ~ 50 milliliters of pure water
Beaker in, stirring, be stored at room temperature 0.5 ~ 1 hour, polymer is by being self-assembly of nanoparticle.
The polymer CF7 of the present invention is used for the near-infrared fluorescence imaging and optical dynamic therapy of tumour cell.
The present invention action principle be:1, improve the solubility of chitosan;2, Cy7 targetings are transported to cancer cell, are used in combination
In near-infrared fluorescence imaging and optical dynamic therapy.
The beneficial effects of the present invention are:
1, polymer of the invention is less than 300 nanometers by the nanoparticle that is self-assembly of, particle diameter, can by intravenously administrable,
Targeting is transported to tumor locus.
2, polymer of the invention and its nanoparticle of formation overcome the defect of chitosan poor solubility, while sharp
It is thin that with the active targeting selective action of tumor cell surface folacin receptor tumour is concentrated on the folic acid on nano-carrier surface
Born of the same parents, but also using the near-infrared fluorescence imaging and optical dynamic therapy of the Cy7 molecules progress tumour cell in nanoparticle.
Brief description of the drawings
Fig. 1 embodiments 1, Cs-N3, C7 and CF7 prepared by embodiment 2 infared spectrum.
Fig. 2 embodiments 1, Cs-N3, CF7 and CF prepared by embodiment 3 uv-spectrogram.
Fig. 3 embodiments 1, Cs-N3, CF7 and C7 prepared by embodiment 2 fluorescence pattern.
Fig. 4 embodiments 4, nanoparticle CF7Ns and C7Ns prepared by embodiment 5 co-focusing imaging figure.
Fig. 5 embodiments 4, nanoparticle CF7Ns and C7Ns prepared by embodiment 5 vitro cytotoxicity.
Embodiment
Below, the present invention will be further described by embodiment, but invention is not limited to these embodiments,
In the scope illustrated by the claims in the present invention, various changes or equivalent substitution can be carried out.
Chitosan 1 is purchased from Shanghai Bai Ao bio tech ltd, and molecular weight is 60 kilodaltons, and deacetylation is 90%.
Embodiment 1
Polymer CF7 synthesis:
Step a:Weigh 800 mg chitosans to be dissolved in 60 mL dry DMFs, be subsequently added 1.6 g phthalic anhydrides, nitrogen is protected
Shield, 120 DEG C of oil bath agitating and heatings.When reaction solution, which becomes, to be clarified, terminating reaction.Reaction solution is poured into appropriate frozen water, separated out white
Color is precipitated.Suction filtration, solid is washed 3 times respectively with ether, acetone, removes unnecessary phthalic anhydride, dry product 2.
Step b:100 mg products 2 are weighed, 10 mL 1-METHYLPYRROLIDONEs are added(NMP), heating stirring dissolving.When
It is placed in after solution cooling in frozen water, adds 616 mg N- bromo-succinimides(NBS), 902 mg triphenylphosphines(TPP).Nitrogen
Gas shielded lower 80 DEG C of two hours of reaction.After reaction terminates, pour the mixture into 100 mL ethanol, separate out solid.By from
The heart(5000 r/min), product is collected, and respectively cleaned three times with ethanol, acetone.Brown solid 3 is obtained after drying.
Step c:Weigh 50 mg products 3 and be dissolved in 5 mL 1-METHYLPYRROLIDONEs, add 50 mg sodium azide
(NaN3), nitrogen protection, agitating and heating 4 hours at 80 DEG C.After reaction terminates, reaction solution is poured in 50 mL ethanol, separated out
Solid.Pass through centrifugation(8000 r/min)Product is collected, product is successively respectively washed three times with ethanol, secondary water, acetone.After drying
Obtain brown solid 4(Cs-N3).Analyzed by infrared spectrum, 6 bromines are replaced by azido on product 3.Pass through infrared spectrum point
Analysis, as shown in figure 1, product 4(Cs-N3)In 2100 cm-1There is infrared absorption peak, show that azido successfully replaces 6 bromines.
Step d:20mg products 4 are weighed, 5 mL dimethyl sulfoxides are dissolved in, 10 mg ALK-FA, 10 mg ALK-Cy7 are added.
Flask rubber stopper seal, after vacuumizing, is protected with nitrogen.2.5 mg cupric sulfate pentahydrates are first added dropwise toward flask with 1 mL syringes
(It is dissolved in 100 μ L secondary waters), it is rear that 2 mg sodium ascorbates are added dropwise(It is dissolved in 100 μ L secondary waters).Reactant is at 50 DEG C
Under, lucifuge reaction 72h.Reaction terminate after by reaction solution specification for 14000 bag filter dialyse 72h.After dialysis, by bag filter
In solid freeze.Analyzed by infrared spectrum, 6 azidos react with the alkynyl in ALK-FA and ALK-Cy7 in product 4,
Generate triazole ring.Analyzed by infrared spectrum, as shown in figure 1, CF7 is in 2100 cm-1There is no infrared absorption peak, show azido
Succeed and alkynyl reaction generation triazole ring.And in 1531 cm-1、1638 cm-1Have two absworption peaks, show folic acid into
Work(is connected on chitosan skeleton.Product is dissolved in dimethyl sulfoxide (DMSO) and surveys UV absorption, as shown in Fig. 2 CF7 is at 280nm
There is UV absorption, show that folic acid has been successfully connected on chitosan skeleton.Product is dissolved in dimethyl sulfoxide (DMSO), excitation wavelength
633nm, surveys its fluorescence intensity.As shown in figure 3, CF7 has ALK-Cy7 characteristic peak at 801nm, show ALK-Cy7 also into
Work(is connected on chitosan skeleton.
Embodiment 2
Chitosan-Cy7 polymer(C7)Synthesis:
The product 4 of 10mg embodiments 1 is weighed, 5 mL dimethyl sulfoxides are dissolved in, 10 mg ALK-Cy7 are added.Flask is close with rubber stopper
Envelope, after vacuumizing, is protected with nitrogen.2.5 mg cupric sulfate pentahydrates are first added dropwise toward flask with 1 mL syringes(It is dissolved in 100 μ L bis-
In secondary water), it is rear that 2 mg sodium ascorbates are added dropwise(It is dissolved in 100 μ L secondary waters).Reactant is at 50 DEG C, lucifuge reaction 72h.
Reaction terminate after by reaction solution specification for 14000 bag filter dialyse 72h.After dialysis, product is freezed.Pass through infrared spectrum
6 azidos react with the alkynyl in ALK-Cy7 in analysis, product 4, generate triazole ring.As shown in figure 1, chitosan-Cy7 is poly-
Compound(C7)In 2100 cm-1There is no infrared absorption peak, show that azido has succeeded to react with the alkynyl in ALK-Cy7 and generate
Triazole ring.Product is dissolved in dimethyl sulfoxide (DMSO), excitation wavelength 633nm surveys its fluorescence spectrum.As shown in figure 3, CF7 is at 801nm
There is ALK-Cy7 characteristic peak, show that ALK-Cy7 has also been successfully connected on chitosan skeleton.
Embodiment 3
Chitosan-FA polymer(CF)Synthesis:
The product 4 of 10mg embodiments 1 is weighed, 5 mL dimethyl sulfoxides are dissolved in, 10 mg ALK-FA are added.Flask is close with rubber stopper
Envelope, after vacuumizing, is protected with nitrogen.2.5 mg cupric sulfate pentahydrates are first added dropwise toward flask with 1 mL syringes(It is dissolved in 100 μ L bis-
In secondary water), it is rear that 2 mg sodium ascorbates are added dropwise(It is dissolved in 100 μ L secondary waters).Reactant is at 50 DEG C, lucifuge reaction 72h.
Reaction terminate after by reaction solution specification for 14000 bag filter dialyse 72h.After dialysis, product is freezed.Pass through infrared spectrum
6 azidos react with the alkynyl in ALK-FA in analysis, product 4, generate triazole ring.As shown in figure 1, chitosan-FA polymerize
Thing(CF)In 2100 cm-1There is no infrared absorption peak, show that azido has succeeded and the alkynyl reaction generation triazole in ALK-FA
Ring.And in 1531 cm-1、1638 cm-1There are two absworption peaks, this shows that ALK-FA is successfully connected on chitosan skeleton.
Product is dissolved in dimethyl sulfoxide (DMSO) and surveys UV absorption, as shown in Fig. 2 CF7 has UV absorption at 280nm, shows ALK-FA
It is successfully connected on chitosan skeleton.
Embodiment 4
Polymer CF7 is used for the preparation method of medicament nano granule:
CF7 made from embodiment 1 is dissolved in dimethyl sulfoxide (DMSO), it is slowly then added dropwise to the burning equipped with pure water with syringe
In cup, stirring mixing is stored at room temperature.The polymer is by being self-assembly of nanoparticle.Concretely comprise the following steps:By CF7 dimethyl
Sulfoxide is made into 0.1 ~ 1 mg/ml solution, then draws 1 milliliter with syringe, it is slowly added dropwise to equipped with 20 ~ 50 milliliters
In the beaker of pure water, stirring is stored at room temperature 0.5 ~ 1 hour, polymer is by being self-assembly of nanoparticle(CF7Ns).
Embodiment 5
Chitosan-Cy7 polymer(C7)Preparation method for medicament nano granule:
C7 is dissolved in dimethyl sulfoxide (DMSO), then it is slowly added dropwise in the beaker equipped with pure water with syringe, stirring mixing,
It is stored at room temperature.The polymer is by being self-assembly of nanoparticle.Concretely comprise the following steps:C7 is made into 0.1 ~ 1 milli with dimethyl sulfoxide (DMSO)
Grams per milliliter solution, then draws 1 milliliter with syringe, it is slowly added dropwise in the beaker equipped with 20 ~ 50 milliliters of pure water, stirred
Mix, be stored at room temperature 0.5 ~ 1 hour, polymer is by being self-assembly of nanoparticle(C7Ns).
Embodiment 6
With Human cervical cancer cell lines Hela cells(Folacin receptor overexpressing cell)With human hepatoma cell line HepG2's cell(Folic acid
Acceptor low expression)For test cell system(Cell is purchased from Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences' cell resource center).
Cell culture processes:Hela cell conservation pipes are taken out from liquid nitrogen container, flash melt thaws in 37 DEG C of water-baths,
Then 1000 rpm centrifuge 5 min, and supernatant is abandoned in suction, take 1 mL DMEM complete culture solutions that cell precipitation is blown and beaten to uniform, transfer
Cause that culture medium is 4 mL in bottle into blake bottle, be placed in 37 DEG C, 5%(v/v)CO2Cultivated in incubator.Take out in liquid nitrogen and freeze
The HepG2 cells deposited, are thawed in 37 DEG C of water, and cell suspension is moved into 1.5 mL centrifuge tubes, 1000 rpm centrifugations 5
Min, abandoning supernatant adds the complete culture solutions of 1 mL RPMI 1640, gently blows and beats uniformly, cell suspension transfer is dealt into
In blake bottle, the complete culture solutions of 3 mL RPMI 1640 are added, blake bottle is placed in 5%(v/v)CO2, train in 37 DEG C of incubators
Support.
Co-focusing imaging is tested:It will be taped against after Hela cells and HepG2 cell dissociations in 24 orifice plates, overnight, cell is complete
After adherent, PBS wash 2 times, respectively the CF7Ns and C7Ns with 20 μ g/mL embodiments 4 and embodiment 54 h are incubated at 37 DEG C.
Then washed with PBS 2 times, fixed 10min is incubated with 4wt% paraformaldehydes, PBS is washed 2 times, then adds DAPI, room temperature lucifuge
10min is incubated, then PBS is washed 2 times.Laser confocal imaging.
Nanoparticle co-focusing imaging result is as shown in Figure 4.Figure 4, it is seen that in Hela cells, CF7Ns's is glimmering
Light strength ratio C7Ns is strong, and in HepG2 cells, CF7Ns fluorescence intensity and C7Ns effects quite, show that the modification of folic acid can
Increase folacin receptor and be overexpressed intake of the tumor cell line to Nano medication.And CF7Ns can be targetted and is transported to folacin receptor
The cell line imaging of height expression, effective means are provided for oncotherapy.
Embodiment 7
With Human cervical cancer cell lines Hela cells(Folacin receptor overexpressing cell)With human hepatoma cell line HepG2's cell(Folic acid
Acceptor low expression)For test cell system(Cell is purchased from Shanghai Life Sciences Research Institute, Chinese Academy Of Sciences' cell resource center).
Cell culture processes:Hela cell conservation pipes are taken out from liquid nitrogen container, flash melt thaws in 37 DEG C of water-baths,
Then 1000 rpm centrifuge 5 min, and supernatant is abandoned in suction, take 1 mL DMEM complete culture solutions that cell precipitation is blown and beaten to uniform, transfer
Cause that culture medium is 4 mL in bottle into blake bottle, be placed in 37 DEG C, 5%(v/v) CO2Cultivated in incubator.Take out in liquid nitrogen and freeze
The HepG2 cells deposited, are thawed in 37 DEG C of water, and cell suspension is moved into 1.5 mL centrifuge tubes, 1000 rpm centrifugations 5
Min, abandoning supernatant adds the complete culture solutions of 1 mL RPMI 1640, gently blows and beats uniformly, cell suspension transfer is dealt into
In blake bottle, the complete culture solutions of 3 mL RPMI 1640 are added, blake bottle is placed in 5%(v/v)CO2, train in 37 DEG C of incubators
Support.
Cytotoxicity experiment:Choose logarithmic phase growth and Hela or HepG2 cells in good condition are through Trypsin Induced
Afterwards so that cell concentration is 0.5-1 × 105Individual/mL, is configured to cell suspension.It is inoculated with by the amount of every μ L cell suspensions of hole 100
To 96 orifice plates, cultivate after 24h, be separately added into 40 μ g/mL Cs-N in embodiment 13, 40 μ g/mL embodiment 4 and embodiment 5
CF7Ns and CFNs, separately set solvent control group and blank control group, in order to investigate optical dynamic therapy effect, the present invention will be added
CF7Ns and CFNs experimental group is divided into four groups, has two groups to use Infrared irradiation(NIR), two groups without Infrared irradiation in addition.Incubate
Educate after 24h, old culture medium is abandoned in suction, and PBS is washed 3 times, and add in every hole 90 μ L serum-frees, without 1640 phenol red culture mediums and
10 μ L MTT solution, continue to be incubated after 4 h, and careful inhale abandons supernatant, and 150 μ L dimethyl sulfoxide (DMSO)s, lucifuge are added in every hole
10min is vibrated, makes bluish violet crystallization all dissolvings, determines the trap in each hole at 570 nm wavelength with multi-function microplate reader,
And the survival rate of cell is calculated as follows.Survival rate(%)=(Experimental group absorption value-solvent control group absorption value)/(Blank group is inhaled
Receipts value-solvent control group absorption value).
The cytotoxicity result of nanoparticle is as shown in Fig. 5.As can be seen that Cs-N from Fig. 53To two kinds of cytotoxicities compared with
Small, CF7Ns and C7Ns can kill cell to varying degrees.In Hela cells, CF7Ns toxicity compares C7Ns
By force, when cell is exposed under infrared lamp, CF7Ns+NIR(Infrared irradiation)Strong toxicity of the toxicity than CF7Ns, C7Ns+
The strong toxicity of NIR toxicity also than C7Ns;And in HepG2 cells, C7Ns is suitable with CF7Ns toxicity, CF7Ns+NIR's
Toxicity and C7Ns+NIR are suitable.This shows that the modification of folic acid can increase the poison that Nano medication is overexpressed tumour cell to folacin receptor
Property, so as to improve antitumor selectivity.Also indicate that optical dynamic therapy can strengthen antitumous effect simultaneously.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (7)
1. a kind of folic acid with tumor-targeting-chitosan-Cy7 polymer, it is characterised in that:Structural formula is as follows:
;
Wherein n is the number of chitosan derivatives repeat unit.
2. folic acid according to claim 1-chitosan-Cy7 polymer, it is characterised in that:The folic acid-chitosan-Cy7
The molecular weight of polymer is 100-1000 kilodaltons.
3. a kind of method for preparing folic acid as claimed in claim 1-chitosan-Cy7 polymer, it is characterised in that:Reaction equation
It is as follows:
;
Wherein n is the number of chitosan derivatives repeat unit;
Concretely comprise the following steps:
Step a:Chitosan 1 is weighed, with phthalic anhydride substituted-amino, N- O-phthalic imido grpups chitosan 2 is obtained;
Step b:6 hydroxyls on product 2 are subjected to bromine substitution reaction, product 3 is obtained;
Step c:6 bromines on product 3 are subjected to azido substitution reaction, product 4 is obtained;
Step d:By product 4 and alkynyl-modified folic acid ALK-FA and alkynyl-modified heptamethine cyanine ALK-Cy7 in nothing
Click reactions are carried out under the catalytic action of brochanite and vitamin C sodium salt, product 5 is obtained;Wherein ALK-FA be by folic acid and
Propargylamine reaction is obtained, and ALK-Cy7 is to be obtained by phenylhydrazine and 3- methyl -2- butanone by series reaction.
4. the preparation method of folic acid according to claim 3-chitosan-Cy7 polymer, it is characterised in that:The shell gathers
The weight average molecular weight of sugar 1 is 10-1000 kilodaltons.
5. the preparation method of folic acid according to claim 3-chitosan-Cy7 polymer, it is characterised in that:The He of product 4
ALK-FA and ALK-Cy7 mass ratio is 2: 1: 1.
6. the medicament nano granule that a kind of folic acid as claimed in claim 1-chitosan-Cy7 polymer is made.
7. medicament nano granule according to claim 6, it is characterised in that:Its preparation method comprises the following steps:Will be described
Folic acid-chitosan-Cy7 polymer 0.1 ~ 1 mg/ml solution is made into dimethyl sulfoxide (DMSO), then with syringe draw 1 milli
Rise, it is added dropwise in the beaker equipped with 20 ~ 50 milliliters of pure water according to the speed of per second one drop, stirs, is stored at room temperature 0.5 ~ 1 small
When, polymer is by being self-assembly of nanoparticle.
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