CN109180680A - A kind of ultraviolet light triggering cross-linking type near-infrared molecular probe and the preparation method and application thereof - Google Patents
A kind of ultraviolet light triggering cross-linking type near-infrared molecular probe and the preparation method and application thereof Download PDFInfo
- Publication number
- CN109180680A CN109180680A CN201810867119.7A CN201810867119A CN109180680A CN 109180680 A CN109180680 A CN 109180680A CN 201810867119 A CN201810867119 A CN 201810867119A CN 109180680 A CN109180680 A CN 109180680A
- Authority
- CN
- China
- Prior art keywords
- compound
- probe
- molecular probe
- ultraviolet light
- type near
- 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.)
- Granted
Links
- 238000004132 cross linking Methods 0.000 title claims abstract description 60
- 239000003068 molecular probe Substances 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 64
- 239000000523 sample Substances 0.000 claims abstract description 52
- 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 claims description 31
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- 150000001875 compounds Chemical class 0.000 claims description 22
- 230000000903 blocking effect Effects 0.000 claims description 18
- 229940125904 compound 1 Drugs 0.000 claims description 17
- 229940125782 compound 2 Drugs 0.000 claims description 17
- 229940126214 compound 3 Drugs 0.000 claims description 17
- 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 claims description 16
- 229940125898 compound 5 Drugs 0.000 claims description 16
- 235000019152 folic acid Nutrition 0.000 claims description 16
- 229960000304 folic acid Drugs 0.000 claims description 16
- 239000011724 folic acid Substances 0.000 claims description 16
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 229940045713 antineoplastic alkylating drug ethylene imines Drugs 0.000 claims description 10
- JKANAVGODYYCQF-UHFFFAOYSA-N prop-2-yn-1-amine Chemical compound NCC#C JKANAVGODYYCQF-UHFFFAOYSA-N 0.000 claims description 10
- 150000001408 amides Chemical group 0.000 claims description 9
- 150000003053 piperidines Chemical class 0.000 claims description 6
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 6
- 229960005055 sodium ascorbate Drugs 0.000 claims description 6
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 6
- 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 6
- 230000001225 therapeutic effect Effects 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 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 claims description 4
- 229960004657 indocyanine green Drugs 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims 2
- 239000012046 mixed solvent Substances 0.000 claims 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims 1
- 239000004472 Lysine Substances 0.000 claims 1
- 230000014759 maintenance of location Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 24
- 210000004881 tumor cell Anatomy 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 8
- 230000003834 intracellular effect Effects 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract 1
- 230000010354 integration Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethyl sulfoxide Natural products CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 17
- 239000000975 dye Substances 0.000 description 16
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 15
- 238000003384 imaging method Methods 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 230000001678 irradiating effect Effects 0.000 description 9
- 238000011580 nude mouse model Methods 0.000 description 9
- 241000699660 Mus musculus Species 0.000 description 7
- 238000000799 fluorescence microscopy Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000007626 photothermal therapy Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 5
- 239000000543 intermediate Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- GRAVJJAQKJDGPM-UHFFFAOYSA-N 3-[2-[7-[3-(2-carboxyethyl)-1,1-dimethylbenzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]propanoic acid;bromide Chemical compound [Br-].OC(=O)CCN1C2=CC=C3C=CC=CC3=C2C(C)(C)\C1=C/C=C/C=C/C=C/C1=[N+](CCC(O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C GRAVJJAQKJDGPM-UHFFFAOYSA-N 0.000 description 4
- 240000002853 Nelumbo nucifera Species 0.000 description 4
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 4
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 description 3
- 230000002687 intercalation Effects 0.000 description 3
- 238000009830 intercalation Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 235000002639 sodium chloride Nutrition 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- YXHKONLOYHBTNS-UHFFFAOYSA-N Diazomethane Chemical compound C=[N+]=[N-] YXHKONLOYHBTNS-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 230000036647 reaction Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229960002668 sodium chloride Drugs 0.000 description 2
- 231100000440 toxicity profile Toxicity 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229920002334 Spandex Polymers 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 208000030270 breast disease Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000571 coke Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000004845 diazirines Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- -1 folic acid compound Chemical class 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000144 sodium(I) superoxide Inorganic materials 0.000 description 1
- 239000004759 spandex Substances 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
-
- 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
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
- C09B23/086—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines more than five >CH- groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of photo-crosslinking type near-infrared molecular probes and the preparation method and application thereof.Specifically, preparation method of the invention is the following steps are included: building, synthesis photo-crosslinking type near-infrared molecular probe DACF；Photo-crosslinking type near-infrared molecular probe DACF is crosslinked in the cell under ultraviolet light triggering.Probe of the invention is irradiated through the light of specific wavelength, the Cabbeen intermediate for generating high activity is bonded with intracellular biological molecule covalent and closes, significantly increases the intake of probe in the cell, and its residence time is extended, it can be realized the long-time tracking to tumour cell.Meanwhile using the photo-thermal effect of probe, tumour cell can be preferably killed.The photo-crosslinking type near-infrared molecular probe that preparation method through the invention obtains can effectively improve enriching quantity and residence time of the probe in vivo tumor, realize the diagnosis and treatment integration of tumour, have important scientific research and economic value.
Description
Technical field
The invention belongs to nir dye functional modification technical fields, and in particular to a kind of double a word used for translations third that ultraviolet light mediates
The preparation method that traditional nir dye Cypate modifies in piperidinyl group, the molecular probe prepared by this method and the spy
Needle is in the near-infrared fluorescence imaging for preparing tumour and is based on the antitumor of photo-thermal therapy (photothermal therapy, PTT)
Application in drug.
Background technique
It is well known that organic near infrared fluorescent dye has good fluorescence quantum yield, lower bio-toxicity, plastic
Molecular structure, lower-price characteristic, be widely used in near-infrared bio-imaging.
Currently, most common and for the nir dye of tumor imaging be indocyanine green by FDA approval
(Indocyanine Green, ICG) can generate near-infrared fluorescent, carry out near-infrared fluorescence imaging, can also be by the light of absorption
It can be converted into heat, increase local temperature, tumour cell, i.e. photo-thermal therapy are killed, to realize the one of tumor diagnosis and therapy
Body.Although lot of documents is reported, can be effectively targeted to based on near-infrared fluorescent Small-molecule probe synthesized by such dyestuff
Tumour, and be used for tumor imaging and photo-thermal therapy, but due to itself being easy to be excluded by cell or tissue metabolism, cause its
The intracorporal blood circulation period is very short, has seriously affected imaging and photo-thermal therapy effect to tumour.
Therefore, developing one kind can promote probe to be enriched in tumor locus and extend the new strategy and new method of residence time
It is significant to raising tumor imaging and therapeutic effect.
Summary of the invention
In order to overcome above-mentioned problems of the prior art, the present invention constructs a kind of photo-crosslinking type near-infrared molecule spy
Needle triggers cross-linking reaction using light, improves enriching quantity and extension its residence time of the probe in tumour, and then effectively improve
The imaging and therapeutic effect of tumour；This method is suitable for a variety of nir dyes, to improve near-infrared molecular probe in tumour
Tracking provides new strategy and means with therapeutic effect for a long time.
The invention adopts the following technical scheme:
A kind of ultraviolet light triggering cross-linking type near-infrared molecular probe, the ultraviolet light triggering cross-linking type near-infrared molecular probe have
Following chemical structural formula:
。
Above-mentioned ultraviolet light triggering cross-linking type near-infrared molecular probe is preparing answering in diagnosing tumor and/or therapeutic reagent
With.
The preparation method of above-mentioned ultraviolet light triggering cross-linking type near-infrared molecular probe, comprising the following steps:
(1) N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys and propargylamine occur it is amide condensed react, obtain compound
1；
(2) compound 1 sloughs blocking group and obtains compound 2；
(3) compound 2 is reacted with the NHS double ethylene imines activated, obtains compound 3；
(4) compound 3 sloughs blocking group and obtains compound 4；
(5) compound 4 is reacted with nir dye, obtains compound 5；
(6) compound 5 is reacted with folic acid, obtains ultraviolet light triggering cross-linking type near-infrared molecular probe.
A kind of preparation method of acid-sensitive nir dye, comprising the following steps:
(1) N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys and propargylamine occur it is amide condensed react, obtain compound
1；
(2) compound 1 sloughs blocking group and obtains compound 2；
(3) compound 2 is reacted with the NHS double ethylene imines activated, obtains compound 3；
(4) compound 3 sloughs blocking group and obtains compound 4；
(5) compound 4 is reacted with nir dye, obtains acid-sensitive nir dye.
A method of extending near-infrared molecular probe residence time in tumour, comprising the following steps:
(1) N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys and propargylamine occur it is amide condensed react, obtain compound
1；
(2) compound 1 sloughs blocking group and obtains compound 2；
(3) compound 2 is reacted with the NHS double ethylene imines activated, obtains compound 3；
(4) compound 3 sloughs blocking group and obtains compound 4；
(5) compound 4 is reacted with nir dye, obtains compound 5；
(6) compound 5 is reacted with folic acid, obtains ultraviolet light triggering cross-linking type near-infrared molecular probe；
(7) it by ultraviolet light triggering cross-linking type near-infrared molecular probe after tumor locus enrichment, through ultraviolet light, completes close red
The extension of outer molecular probe residence time in tumour.
In above-mentioned technical proposal, ultraviolet light triggering cross-linking type near-infrared molecular probe is dissolved in PBS(phosphate-buffered salt, pH=
7.2~7.4) it (concentration is 45 μM) in injection, injects in lotus knurl body, probe is then waited to reach maximum rich in tumor locus
Collection amount completes near-infrared molecular probe in the extension of tumor locus residence time after ultraviolet light.
In above-mentioned technical proposal, in step (1), N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys and propargylamine
Molar ratio be 1: 1；Amide condensed reaction carries out in the presence of HOBt, HBTU and diisopropylethylamine；Amide condensed reaction is
Room temperature reaction 15~24 hours.
In above-mentioned technical proposal, in step (2), it is molten in methylene chloride/trifluoroacetic acid mixing that compound 1 sloughs blocking group
It is carried out in agent；Methylene chloride, trifluoroacetic acid volume ratio be 4: 1.
In above-mentioned technical proposal, in step (3), the reaction carries out in the presence of diisopropylethylamine；Compound 2, NHS
Double ethylene imines of activation, the molar ratio of diisopropylethylamine are 1: 1: 2.
In above-mentioned technical proposal, in step (4), it is mixed in n,N-Dimethylformamide/piperidines that compound 3 sloughs blocking group
It is carried out in bonding solvent；N,N-dimethylformamide, piperidines volume ratio be 4: 1.
In above-mentioned technical proposal, in step (5), the molar ratio of compound 4 and nir dye is 1.2: 1；It is described close red
Outer dyestuff is indocyanine green carboxy derivatives Cypate.
In above-mentioned technical proposal, in step (6), the folic acid is the folic acid of nitrine modification；Compound 5 is reacted with folic acid
It is carried out in the presence of sodium ascorbate and copper sulphate；Compound 5, folic acid, sodium ascorbate, copper sulphate molar ratio be 1: 1:
0.1∶0.05。
In above-mentioned technical proposal, in step (7), when ultraviolet light, wavelength is 365~405 nm, exposure intensity is 1~
12 W/cm2, irradiation time is 1~10 minute；Probe is 0.5~1 hour in the enrichment time of tumor locus.
In above-mentioned technical proposal, compound 1, compound 2, compound 3, compound 4, compound 5 chemical structural formula point
It is not as follows:
The chemical structural formula of double ethylene imines of NHS activation is as follows:
The chemical structural formula of the folic acid of nitrine modification is as follows:
Specifically, method provided by the invention comprising following steps:
(1) it constructs, synthesize photo-crosslinking type near-infrared molecular probe:
According to the synthesis step of design: acyl occurs for propargylamine and N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys first
Amine condensation reaction, then with 20% trifluoroacetic acid (methylene chloride: trifluoroacetic acid=4:1, v/v)；By midbody compound Boc
Blocking group is taken off；Then it with is reacted with the activated diazirine of NHS, resulting midbody compound uses 20% again
Piperidines (n,N-Dimethylformamide: piperidines=4:1, v/v) sloughs blocking group Fmoc, occurs with nir dye Cypate
Further under sodium ascorbate and catalysis of cupric sulphate, with folic acid point occurs for amide condensed reaction, obtained midbody compound
It hits chemical reaction and obtains final probe DACF, structural formula is as follows:
(2) photo-crosslinking effect of the photo-crosslinking type near-infrared molecular probe DACF after ultraviolet light in the cell:
The photo-crosslinking type near-infrared molecular probe obtained in step (1) is dissolved in cell culture medium, it is thin to be added to 4T1
In born of the same parents' culture dish (concentration: 1 μM), it is put into 12 h of incubator culture.It is clear with PBS to the end of probe and 4T1 cell incubation time
It washes one time, after fresh culture medium is added, is placed under the ultraviolet light that wavelength is 365 ~ 405 nm and irradiates, exposure intensity is 1 ~ 12
W/cm2, irradiation time is 1 ~ 10 minute, and molecular probe can be crosslinked with macromolecular compounds such as albumen in tumour cell
Reaction is advantageously implemented the long-time tracking to tumour cell to extend its residence time in tumour cell.
(3) photo-crosslinking effect of the photo-crosslinking type near-infrared molecular probe DACF after ultraviolet light in tumour:
The photo-crosslinking type near-infrared molecular probe obtained in step (1) is dissolved in PBS injection (concentration: 45 μM),
By probe injection lotus, there are two in the BALB/c/nu Female nude mice body of tumor (4T1 breast cancer) in a manner of tail vein injection.Wait visit
Needle is placed under the ultraviolet light that wavelength is 365 ~ 405 nm and irradiates after tumor locus is enriched with 0.5 ~ 1 hour, and exposure intensity is 1 ~ 12
W/cm2, irradiation time is 1 ~ 10 minute, and molecular probe can be crosslinked with macromolecular compounds such as albumen in tumour cell
Reaction is advantageously implemented the long-time tracking diagnosis to tumor locus to extend its residence time in tumour cell.
In the present invention, after compound 5 is reacted with folic acid, using semipreparative high performance liquid chromatography separating-purifying, purple is obtained
Outer light triggers cross-linking type near-infrared molecular probe, and product is the solid powder of cyanic colours.Preferably, the high performance liquid chromatography
Separation method are as follows: C18 column, 3.5 μm, 4.6 × 100 mm；Mobile phase: A is trifluoroacetic acid: water=1:1000；B is trifluoro second
Acid: acetonitrile=1:1000；Flow velocity: 1 mL/min；Linear gradient elution program: 0 min, A:B=95:5；12 min, A:B=
0:100.
In the present invention, the ultraviolet light of ultraviolet light is provided by ultraviolet light emission device, and ultraviolet light emission device is hand-held
Formula ultraviolet lamp or ultraviolet laser, preferably ultraviolet light emission device are ultraviolet laser.
Preparation method of the invention triggers cross-linking reaction by light, improves enriching quantity and extension of the probe in tumour cell
Its residence time.
Due to the application of the above technical scheme, the invention has the following advantages over the prior art:
(1) double ethylene imines (diazirine) functional modification nir dye, light trigger condition temperature have been used for the first time in the present invention
With it is simple；
(2) photo-crosslinking type near-infrared molecular probe issues third contact of a total solar or lunar eclipse cross-linking reaction in ultraviolet light triggering and is not affected by the external environment；
(3) after probe enters tumour cell, under the irradiation of ultraviolet light, it can be quickly converted to Cabbeen reactive intermediate, then
Covalently cross-linked on the high molecular weight protein of surrounding by the way that intercalation reaction quickly occurs with C-H, O-H, N-H and S-H, crosslinking is anti-
It should very efficiently.
Detailed description of the invention
Fig. 1 is the synthetic schemes of photo-crosslinking type near-infrared molecular probe DACF in embodiment 1；
Fig. 2 is the mechanism of action schematic diagram of photo-crosslinking type near-infrared molecular probe DACF in embodiment 2；
The high-efficient liquid phase color spectral purity that Fig. 3 is photo-crosslinking type near-infrared molecular probe DACF in embodiment 3 characterizes (a) and high score
Distinguish mass spectral characteristi (b)；
Fig. 4 is the ultra-violet absorption spectrum and fluorescence spectrum situation (a) of photo-crosslinking type near-infrared molecular probe DACF in embodiment 4
And the toxicity profile (b) of probe；
Fig. 5 is the survival fluorescence photo of 4T1 cell after irradiating 405 nm laser of different time in embodiment 5；
Fig. 6 is the fluorescence photo (a) of 4T1 cell and corresponding standard in different time after irradiating 405 nm laser in embodiment 6
Fluorescence intensity figure (b)；
Fig. 7 is the near-infrared fluorescent photo (a) of mouse tumor locus in different time after irradiating 405 nm laser in embodiment 7
With corresponding standard fluorescence signal strength map (b)；
Fig. 8 is the optoacoustic photo (a) of mouse tumor locus and correspondence in different time after irradiating 405 nm laser in embodiment 8
Standard photoacoustic signal value (b).
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further explained.It should be understood that these embodiments
The technical solution being only used in the explanation and illustration present invention, and it is not intended to limit the scope of the present invention.In addition, unless otherwise saying
Bright, material used in the following example, reagent, instrument etc. can be obtained by commercial means.
Embodiment 1: the synthesis and characterization of photo-crosslinking type near-infrared molecular probe
(1) propargylamine (0.29 mL), N,N-dimethylformamide (30 mL), HOBt are added in 100 mL round-bottomed flasks
(0.68 g, 5.04 mmol), HBTU(1.91 g, 5.04 mmol) and diisopropylethylamine (1.08 g, 8.4 mmol) is no
It is disconnected to stir evenly.Then, into reaction flask be added N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys (1.97 g, 4.2
Mmol), continue to stir, react at room temperature overnight (20 hours).After reaction, revolving removes solvent, and 30 mL ethyl acetate are added
Re-dissolve midbody compound.Subsequent organic phase uses 30 mL deionized waters, saturated sodium bicarbonate and sodium-chloride water solution respectively
Respectively wash it is primary, with anhydrous magnesium sulfate it is dry after be spin-dried for, obtain the compound 1 of white powder intermediate 1(its structure as shown in figure 1
It is shown) (1.95 g, yield: 92%).
1H NMR (400 MHz, d 6 - DMSO), δ (ppm): 8.35 (s, 1H), 7.90 (d, J=7.5 Hz, 2H),
7.74 (dd, J=7.2,4.3 Hz, 2H), 7.48 (d, J=8.2 Hz, 1H), 7.42 (t, J=7.4 Hz, 2H),
7.34 (t, J=7.4 Hz, 2H), 6.77 (t, J=5.1 Hz, 1H), 4.25 (m, 3H), 3.95 (Hz of d, J=5.1,
1H), 3.86 (dd, J=5.2,2.3 Hz, 2H), 3.10 (t, J=2.5 Hz, 1H), 2.89 (Hz of d, J=2.7,
2H), 1.56 (dd, J=10.2,4.1 Hz, 2H), 1.37 (s, 11H), 1.23 (s, 2H)；13C NMR (101 MHz,
CDCl3), δ (ppm): 171.02,155.84,143.24,140.82,127.29,126.63,124.59,119.53,78.77,
71.29,66.61,54.15,46.65,31.49,29.16,28.71,27.96,21.94；MS (ESI-TOF) Calc'd
for C29H35N3NaO5 [M+Na]+, 528.2474；Found, 528.2470.
(2) trifluoroacetic acid for the methylene chloride and 4 mL that 16 mL are added in 50 mL round-bottomed flasks stirs evenly.Then
Midbody compound 1(1.01 g, 2 mmol) is added in reaction flask, 1 h of reaction is stirred at room temperature.After reaction, revolving is removed
Dereaction liquid is purified (methylene chloride: methanol=10:1, v/v) with silica gel column chromatography, obtains its knot of white solid intermediate 2(
Shown in the compound 2 of structure as shown in figure 1) (0.65 g, yield: 80%).
1H NMR (400 MHz,d 6 - DMSO), δ (ppm): 8.43 (s, 1H), 7.93 (d, J=7.5 Hz, 2H),
7.82 (s, 2H), 7.77 (t, J=7.2 Hz, 2H), 7.55 (d, J=8.3 Hz, 1H), 7.46 (Hz of t, J=7.3,
2H), 7.37 (t, J=7.4 Hz, 2H), 4.31 (d, J=6.5 Hz, 2H), 4.26 (d, J=6.2 Hz, 1H), 3.99
(dd, J=13.6,8.9 Hz, 1H), 3.90 (dd, J=5.3,2.3 Hz, 2H), 3.15 (t, J=2.4 Hz, 1H),
2.80 (d, J=5.4 Hz, 2H), 1.61 (m, 4H), 1.36 (m, 2H)；13C NMR (101 MHz,d 6 - DMSO), δ
(ppm): 171.69,155.98,43.81,140.70,127.62,127.05,125.29,120.10,81.03,73.02,
65.59,54.22,46.64,31.17,27.94,26.52,22.40；MS (ESI-TOF) Calc'd for C24H27N3NaO3
[M+Na]+, 428.1950；Found, 428.1932.
(3) methylene chloride of 15 mL, double ethylene imine (NHS- of NHS activation are added in 50 mL round-bottomed flasks
Diazirine) (0.25 g, 1 mmol), midbody compound 2(0.41 g, 1 mmol) and diisopropylethylamine (278 μ
L, 2 mmol), 2 h of reaction are stirred at room temperature.After reaction, organic phase is washed three times with 25 mL deionizations, 25 mL sodium chloride
Aqueous solution is washed one time, is spin-dried for after being dried with anhydrous magnesium sulfate.Then purified with silica gel column chromatography (methylene chloride: methanol=20:
1, v/v), obtain shown in the compound 3 of white solid intermediate 3(its structure as shown in figure 1) (0.49 g, yield: 95%).
1H NMR (400 MHz,d 6 - DMSO), δ (ppm): 8.30 (s, 1H), 7.86 (d, J=7.5 Hz, 2H),
7.78 (s, 1H), 7.70 (t, J=7.0 Hz, 2H), 7.43 (d, J=8.2 Hz, 1H), 7.39 (Hz of t, J=7.4,
2H), 7.30 (t, J=7.4 Hz, 2H), 4.21 (dt, J=16.2,6.8 Hz, 3H), 3.90 (dd, J=13.7,8.8
Hz, 1H), 3.82 (d, J=2.9 Hz, 2H), 3.07 (s, 1H), 2.97 (dt, J=14.0,7.0 Hz, 2H), 1.90
(t, J=7.7 Hz, 2H), 1.52 (m, 4H), 1.34 (dd, J=14.1,7.2 Hz, 2H), 1.24 (d, J=39.2
Hz, 2H), 0.93 (s, 3H)；13C NMR (101 MHz,d 6 - DMSO), δ (ppm): 172.22,170.88,156.37,
144.26,141.13,128.06,127.48,125.76,120.52,81.49,73.43,66.05,54.83,47.09,
31.95,30.24,29.13,28.36,19.74；MS (ESI-TOF) Calc'd for C29H33N5NaO4 [M+Na]+,
538.2430；Found, 538.2415.
(4) piperidines for the N,N-dimethylformamide and 2 mL that 8 mL are added in 50 mL round-bottomed flasks stirs evenly.
Then midbody compound 3(0.5 g, 0.97 mmol) is added in reaction flask, 1 h of reaction is stirred at room temperature.After reaction,
Revolving removes reaction solution, is purified (methylene chloride: methanol=10:1, v/v) with silica gel column chromatography, is obtained among yellow oil
Shown in the compound 4 of body 4(its structure as shown in figure 1) (0.2 g, yield: 70%).
1H NMR (400 MHz,d 6 - DMSO), δ (ppm): 8.25 (s, 1H), 7.84 (s, 1H), 3.88 (s, 2H),
3.42 (s, 2H), 3.14 (d, J=7.1 Hz, 1H), 3.11 (m, 1H), 3.03 (dd, J=12.5,6.6 Hz, 2H),
1.97 (t, J=7.7 Hz, 2H), 1.60 (d, J=8.0 Hz, 2H), 1.38 (m, 4H), 1.27 (d, J=5.8
Hz, 2H), 1.01 (s, 3H)；13C NMR (101 MHz,d 6 - DMSO), δ (ppm): 174.99,170.43,81.29,
72.75,54.47,34.71,29.78,28.95,27.73,25.80,22.65,19.29；MS (ESI-TOF) Calc'd for
C14H24N5NaO2 [M+Na]+, 316.1749；Found, 316.1737.
(5) in 20 mL round-bottomed flasks be added HOBt(4.7 mg, 0.035 mmol), 1- (3- dimethylamino-propyl)-
3- ethyl-carbodiimide hydrochloride (6.7 mg, 0.035 mmol) and Cypate(18.1 mg, 0.029 mmol), room temperature is stirred
Mix 20 min of activation.Then to addition midbody compound 4(10.3 mg, 0.035 mmol in reaction flask), continue room temperature and stirs
Mix 8 h of reaction.After reaction, it using semipreparative high performance liquid chromatography separating-purifying, obtains in the solid powder of cyanic colours
Shown in the compound 5 of mesosome 5(its structure as shown in figure 1) (15.1 mg, yield: 58%).
1H NMR (400 MHz, CDCl3), δ (ppm): 11.36 (s, 1H), 9.62 (s, 1H), 8.99 (s, 1H),
8.12 (dd, J=8.3,4.5 Hz, 2H), 7.98 (dd, J=19.3,8.4 Hz, 4H), 7.82 (s, 1H), 7.65
(dt, J=15.5,8.0 Hz, 3H), 7.51 (m, 2H), 7.42 (d, J=9.4 Hz, 1H), 6.59 (s, 1H), 6.31
(s, 1H), 5.46 (s, 5H), 4.59 (dd, J=7.5,5.7 Hz, 1H), 4.44 (m, 2H), 4.00 (d, J=3.6
Hz, 2H), 3.39 (d, J=5.4 Hz, 2H), 3.22 (s, 3H), 2.06 (dd, J=11.4,5.9 Hz, 6H), 1.99
(d, J=3.3 Hz, 6H), 1.71 (m, 2H), 1.46 (d, J=9.2 Hz, 6H), 1.33 (d, J=6.6 Hz, 4H),
1.02 (s, 2H), 0.92 (s, 3H)；HRMS (ESI): m/z calcd for C55H62N7O5 + [M]+, 900.4807；
Found, 899.9542.
(6) DMSO (1 mL), midbody compound 5(9 mg, 0.01 mmol are added in 5 mL round-bottomed flasks) and
The folic acid compound (5.2 mg, 0.01 mmol) for having modified nitrine molecule, stirs evenly.Meanwhile by sodium ascorbate
The mixing of (0.2 mg, 10 mmol%) and anhydrous cupric sulfate (0.09 mg, 5 mmol%) is dissolved in 1 mL deionized water, then will be mixed
It closes liquid to be added in reaction flask, 8 h of reaction is stirred at room temperature.After reaction, it is separated using semipreparative high performance liquid chromatography
Purification, obtains the solid powder of molecular probe DACF(cyanic colours, 12.8 mg, yield: 94%).
HRMS (ESI): m/z calcd for C77H87N18O10 + [M]+, 1423.6847；Found, 1423.1622.
Embodiment 2: photo-crosslinking effect of the photo-crosslinking type near-infrared molecular probe DACF after ultraviolet light
As shown in Fig. 2, photo-crosslinking type near-infrared molecular probe DACF obtained in embodiment 1 is diluted in cell culture medium,
It is added in 4T1 Tissue Culture Dish and is incubated for altogether later.After probe enters tumour cell, through ultraviolet light, probe institute tape base
Group can generate intermediate Cabbeen, then covalently by C-H, O-H, N-H and S-H intercalation reaction and the high molecular weight protein of surrounding etc.
Bond is closed, and connect probe molecule securely with the high molecular weight protein etc. in tumour cell, to extend probe molecule in the cell
Residence time, improve the imaging and therapeutic effect of probe molecule, mechanism is referring to attached drawing 2.
Embodiment 3: the high-efficient liquid phase color spectral purity characterization and high resolution mass spectrum of photo-crosslinking type near-infrared molecular probe DACF
Characterization
By photo-crosslinking type near-infrared molecular probe obtained in embodiment 1 with solvent methanol be diluted to concentration be 5 μM after, pass through
High resolution mass spectrum carries out molecular weight to probe and determines, and carries out purity analysis to it using high performance liquid chromatograph.
As shown in Figure 3a, sample is analyzed using 1260 high performance liquid chromatograph of Agilent, the guarantor of probe DACF
It stays the time at 8.176 minutes, further peak area out is integrated, sample middle probe is calculated at concentrations up to 99%.Fig. 3 b
The theoretical m/z:1423.6847 for showing probe DACF, actually obtains m/z:1423.1622 in high resolution mass spectrum spectrogram, the two phase
It coincide, as wanted compound.
Embodiment 4: the ultra-violet absorption spectrum and fluorescence spectrum situation of photo-crosslinking type near-infrared molecular probe DACF and spy
The toxicity profile of needle
By photo-crosslinking type near-infrared molecular probe obtained in embodiment 1 with solvent be diluted to concentration be 5 μM after, use respectively
Uv-visible absorption spectra instrument and stable state/transient state Fluorescence Spectrometer remove the ultra-violet absorption spectrum and fluorescence spectrum of measurement probe；It is logical
Cross MTT(thiazolyl blue) colorimetric method investigates probe to the cytotoxicity of 4T1.
As shown in fig. 4 a, it is found by ultra-violet absorption spectrum, there are two bright in the range of 650-800 nm by probe DACF
Aobvious absorption peak, maximum absorption band is in 785 nm；It is found by fluorescence spectrum, probe DACF has one within the scope of 805-900 nm
A emission peak, emission maximum is in 830 nm.
Fig. 4 b is the cytotoxicity experiment of molecular probe DACF, the survival rate and concentration and probe concentration of cell there are dependence,
With the increase of concentration, probe constantly increases the toxicity of cell.But molecular probe DACF concentration be 14 μM when,
Cell survival rate still has 80% or more, this concentration is much higher by concentration and probe concentration used in clinic.
Embodiment 5: degree of impairment of the 405 nm lasers to 4T1 cell of different time is irradiated
Cytotoxicity assay: mouse mastopathy cell (4T1) (density 3 × 10 in 6 orifice plates5A/hole) culture, it is incubated for 24
After h, through ultraviolet laser, (405 nm, power are 1 W/cm2) the irradiation different time (0,1,2,4,8,10 min), it is then placed in
Incubator continues to cultivate 4 h, abandons supernatant later, is added Live-Dead dyeing liquor, 30 min of incubator culture, uses Lycra after dye
Fluorescence microscope carries out observation of taking pictures.
As shown in figure 5, when irradiation 405 nm laser time within 4 minutes, compared to it is no irradiation laser contrast groups,
It can clearly be seen that the survival rate of 4T1 cell is very high, not dead cell substantially.However, when the irradiation of 405 nm laser
Time, 4T1 cell just will receive stronger damage at 8 minutes or more, the cell death of large area occurs.Therefore irradiation 405
The time of nm laser preferably 1.5 minutes.
Embodiment 6: photo-crosslinking type near-infrared molecular probe DACF 4T1 into the cell after ultraviolet light probe delay
Situation
Based on the method recorded in embodiment 2, by 4T1 cell and it is inoculated in the copolymerization coke capsule of 8 orifice plates, every hole 5 × 103It is a
Cell is put into incubator and cultivates 24 hours.Culture medium is abandoned later, and (the probe of cell culture medium containing probe of same concentrations is added
1 μM of concentration), 200 μ L are put into 12 h of incubator culture, to the end of probe and 4T1 cell incubation time, in 405 nm laser
Device (1 W/cm2) 1.5 min of irradiation, while experimental comparison group is set, irradiation terminates, and fresh culture medium is added and is placed in culture
Continue to cultivate 8 h, 24 h and 48 h in case.Core is contaminated with 33342 dye liquor of Hoechst afterwards, is existed with confocal laser scanning microscope
Fluorescence after 405 nm laser illuminations in different time in 4T1 cell
It as shown in Figure 6 a, is the same in the intracellular red fluorescence intensity of 4T1 when originating 0 h.Irradiating 405
After nm laser when 8 h, 4T1 intracellular red fluorescence is significantly stronger than the control group for not irradiating 405 nm laser.And with
The very fast of the control group fluorescent weakening of 405 nm laser is not irradiated in the extension of time, and after 24 h, 4T1 is intracellular
It is barely perceivable red fluorescent.The experimental group of comparison 405 nm laser of irradiation, in 48 h, its 4T1 into the cell can be with
Observe apparent red fluorescence.Fig. 6 b is corresponding fluorescent quantitation data, irradiates the experimental group of 405 nm laser, can extend
Probe is detained 48 h in the cell, and the control group for not irradiating 405 nm laser only maintains 24 h in the cell.Therefore light
Cross-linking type near-infrared molecular probe DACF can potential application in Cellular tracking.
Embodiment 7: photo-crosslinking type near-infrared molecular probe DACF is in mouse tumor position near-infrared fluorescence imaging situation
Based on the method recorded in embodiment 2, BALB/c/nu Female nude mice of the lotus there are two tumor is taken, with the side of tail vein injection
Formula injects probe DACF in nude mouse, and administration concentration is 45 μM (200 μ L/ every).After injecting probe half an hour, use
405 nm laser (1W/cm2) to 10 min of tumour laser irradiation on the left of mouse, right side tumor does not irradiate 405 nm laser work then
For control group.Be placed in toy IVIS Lumina XRMS living imaging system, observation probe is swollen at two of nude mice
Distribution situation in tumor, and two tumor locus in different time points glimmering that software calculates nude mice is analyzed by IVIS living imaging
Luminous intensity.
As shown in Figure 7a, in 1 h, fluorescence signal intensity of the molecular probe in two tumours of mouse or so is consistent
's.Later with the extension of time, probe is gradually metabolized in mouse body, in 8 h, the swollen of 405 nm laser is not irradiated on right side
The fluorescence signal at tumor position gradually lowers, and the fluorescence signal of the tumor locus lower than 405 nm laser of left side irradiation.12
When h, the fluorescence signal of the tumor locus of 405 nm laser is not irradiated almost without and the swollen of 405 nm laser is irradiated in left side in right side
Tumor position still has apparent fluorescence signal until 24 h.It is counted by the fluorescence signal of Fig. 7 b tumor locus, it can be clear
See, does not irradiate 405 nm laser tumor locus in 12 h without fluorescence intensity signals, and experimental group tumor locus is glimmering
Optical signal can continue 24 h.Demonstrating photo-crosslinking probe of the invention by the result of internal fluorescence imaging can be in tumour portion
Position carries out photo-crosslinking, is crosslinked in tumour, reduces cell metabolism outlet, is detained probe for a long time in tumor locus, from
And extend the fluorescence imaging time.
Embodiment 8: photo-crosslinking type near-infrared molecular probe DACF is in mouse tumor position photoacoustic imaging situation
BALB/c/nu Female nude mice of the lotus there are two tumor is taken, is injected probe DACF in nude mouse in a manner of tail vein injection,
Administration concentration is 45 μM (200 μ L/ every).After injecting probe half an hour, 405 nm laser (1W/cm are used2) to old
10 min of tumour laser irradiation, right side tumor do not irradiate 405 nm laser then as a control group on the left of mouse.Simultaneously by petty action object light
Acoustic imaging system is opened, to the water temperature in photoacoustic imager sea water bath up to 37oWhen C, it is put into the nude mice anaesthetized, scans nude mice
Two tumor locus images.The photoacoustic imaging data of acquisition are analyzed into software using MSOT inSight/inVision later
Carry out reconstruction analysis.
It can be seen that, when 8 h, the tumor locus of 405 nm laser of irradiation shows very strong optoacoustic from Fig. 8 a
Signal, the tumor locus photoacoustic signal for not irradiating 405 nm laser is very weak, and in 12 h, photoacoustic signal is already close to disappearing
It loses.The tumor locus for then irradiating 405 nm laser still shows clearly photoacoustic signal in 24 h.Pass through Fig. 8 b tumour portion
The photoacoustic signal statistics of position, can also be observed that, irradiate the tumor locus of 405 nm laser until that 24 h still have is stronger
Photoacoustic signal, and control group photoacoustic signal value has leveled off to 0.In conjunction with the above fluorescence imaging and photoacoustic imaging as a result, can be with
It show that probe DACF of the invention can carry out photo-crosslinking in tumor locus, and is crosslinked in tumour, reduce cell metabolism
Outlet is detained probe for a long time in tumor locus, to extend the photoacoustic imaging time.
Research of the photaesthesia probe application in terms of photo-crosslinking phenotypic marker albumen has been achieved for the development to attract people's attention.For
The shortcomings that overcoming traditional near-infrared molecular probe, constructs a kind of photo-crosslinking type near-infrared molecular probe, utilizes light triggering crosslinking
Reaction improves probe in the enriching quantity of tumor locus and extends its residence time, and then effectively improves the imaging effect of tumour.
It has several advantages that firstly, light trigger condition is mild, simple；Second, photo-crosslinking is not by items such as tumor locus pH
The influence of part；Third under the irradiation of ultraviolet light, it is intermediate to be quickly converted to Cabbeen activity after probe enters tumour cell
Body, then by the way that the covalently cross-linked high molecular weight protein in surrounding of intercalation reaction quickly occurs with C-H, O-H, N-H and S-H
On, cross-linking reaction is very efficient.Thus there is near-infrared molecular probe in fields such as biologies further to apply.
Claims (10)
1. a kind of ultraviolet light triggers cross-linking type near-infrared molecular probe, which is characterized in that the ultraviolet light triggering cross-linking type is closely red
Outer molecular probe has following chemical structural formula:
。
2. the triggering cross-linking type near-infrared molecular probe of ultraviolet light described in claim 1 is preparing diagnosing tumor and/or therapeutic reagent
In application.
3. ultraviolet light described in claim 1 triggering cross-linking type near-infrared molecular probe preparation method, which is characterized in that including with
Lower step:
(1) N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys and propargylamine occur it is amide condensed react, obtain compound
1；
(2) compound 1 sloughs blocking group and obtains compound 2；
(3) compound 2 is reacted with the NHS double ethylene imines activated, obtains compound 3；
(4) compound 3 sloughs blocking group and obtains compound 4；
(5) compound 4 is reacted with nir dye, obtains compound 5；
(6) compound 5 is reacted with folic acid, obtains ultraviolet light triggering cross-linking type near-infrared molecular probe.
4. a kind of preparation method of acid-sensitive nir dye, which comprises the following steps:
(1) N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys and propargylamine occur it is amide condensed react, obtain compound
1；
(2) compound 1 sloughs blocking group and obtains compound 2；
(3) compound 2 is reacted with the NHS double ethylene imines activated, obtains compound 3；
(4) compound 3 sloughs blocking group and obtains compound 4；
(5) compound 4 is reacted with nir dye, obtains acid-sensitive nir dye.
5. a kind of method for extending near-infrared molecular probe residence time in tumour, which comprises the following steps:
(1) N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-Lys and propargylamine occur it is amide condensed react, obtain compound
1；
(2) compound 1 sloughs blocking group and obtains compound 2；
(3) compound 2 is reacted with the NHS double ethylene imines activated, obtains compound 3；
(4) compound 3 sloughs blocking group and obtains compound 4；
(5) compound 4 is reacted with nir dye, obtains compound 5；
(6) compound 5 is reacted with folic acid, obtains ultraviolet light triggering cross-linking type near-infrared molecular probe；
(7) it by ultraviolet light triggering cross-linking type near-infrared molecular probe after tumor locus enrichment, through ultraviolet light, completes close red
The extension of outer molecular probe residence time in tumour.
6. according to method described in claim 3,4 or 5, which is characterized in that N- tertbutyloxycarbonyl-N '-fluorenylmethyloxycarbonyl-D-
The molar ratio of lysine and propargylamine is 1: 1；Compound 1 sloughs blocking group in methylene chloride/trifluoroacetic acid in the mixed solvent
It carries out；The molar ratio of compound 2 and double ethylene imines of NHS activation is 1.
7. according to method described in claim 3,4 or 5, which is characterized in that compound 3 sloughs blocking group in N, N- diformazan
Base formamide/piperidines in the mixed solvent carries out；The molar ratio of compound 4 and nir dye is 1.2: 1；The nir dye
For indocyanine green carboxy derivatives.
8. according to method described in claim 3 or 5, which is characterized in that the folic acid is the folic acid of nitrine modification；Compound
5 carry out in the presence of sodium ascorbate and copper sulphate with reacting for folic acid；Compound 5, folic acid, sodium ascorbate, copper sulphate
Molar ratio is 1: 1: 0.1: 0.05.
9. according to the method described in claim 5, it is characterized in that, wavelength is 365~405 nm, irradiation when ultraviolet light
Intensity is 1~12 W/cm2, irradiation time is 1~10 minute.
10. ultraviolet light described in claim 1 triggers cross-linking type near-infrared molecular probe increased retention in preparation tumour
Application in probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810867119.7A CN109180680B (en) | 2018-08-01 | 2018-08-01 | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810867119.7A CN109180680B (en) | 2018-08-01 | 2018-08-01 | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109180680A true CN109180680A (en) | 2019-01-11 |
| CN109180680B CN109180680B (en) | 2020-05-08 |
Family
ID=64920463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810867119.7A Expired - Fee Related CN109180680B (en) | 2018-08-01 | 2018-08-01 | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109180680B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110407873A (en) * | 2019-07-30 | 2019-11-05 | 苏州大学 | A kind of tumor microenvironment H2O2Respond cross-linking type near-infrared molecular probe and its application |
| WO2020024302A1 (en) * | 2018-08-03 | 2020-02-06 | 苏州大学张家港工业技术研究院 | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application therefor |
| CN111803700A (en) * | 2020-07-15 | 2020-10-23 | 广东省医疗器械研究所 | Adhesive and preparation method thereof |
| CN112239604A (en) * | 2020-11-24 | 2021-01-19 | 浙江理工大学 | Reactive disperse dye, preparation method and dyeing application thereof |
| CN112341838A (en) * | 2020-11-24 | 2021-02-09 | 浙江理工大学上虞工业技术研究院有限公司 | Reactive dye, reactive dye ink, and preparation method and application thereof |
| CN112409815A (en) * | 2020-11-24 | 2021-02-26 | 浙江理工大学上虞工业技术研究院有限公司 | Bis-aziridine type disperse dyes having double reactive groups, process for their preparation and dyeing applications |
| CN112457682A (en) * | 2020-11-24 | 2021-03-09 | 浙江理工大学上虞工业技术研究院有限公司 | Disperse dye ink for digital ink-jet printing and preparation method and application thereof |
| CN112521373A (en) * | 2019-09-19 | 2021-03-19 | 南京大学 | Multi-modal probe and preparation method and application thereof |
| CN114149482A (en) * | 2021-12-01 | 2022-03-08 | 苏州大学 | Intelligent conversion dual-stimulation response type probe for chelating metal ions as well as preparation method and application of probe |
| WO2022099565A1 (en) * | 2020-11-12 | 2022-05-19 | 苏州大学 | Red-light-mediated nucleic acid anchoring-type fluorescent probe, and preparation method therefor and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102838671A (en) * | 2011-06-23 | 2012-12-26 | 北京大学 | Growth hormone with site-specific mutagenesis and site-specific decoration, preparation method and applications of growth hormone |
| CN103435648A (en) * | 2013-08-29 | 2013-12-11 | 厦门大学 | Deoxyribonucleic acid (DNA) base analogue with photo-crosslinking group biaziridine and synthetic method thereof |
| CN106929482A (en) * | 2015-12-31 | 2017-07-07 | 北京大学 | Influenza virus, its live vaccine of rite-directed mutagenesis and its preparation method and application |
-
2018
- 2018-08-01 CN CN201810867119.7A patent/CN109180680B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102838671A (en) * | 2011-06-23 | 2012-12-26 | 北京大学 | Growth hormone with site-specific mutagenesis and site-specific decoration, preparation method and applications of growth hormone |
| CN103435648A (en) * | 2013-08-29 | 2013-12-11 | 厦门大学 | Deoxyribonucleic acid (DNA) base analogue with photo-crosslinking group biaziridine and synthetic method thereof |
| CN106929482A (en) * | 2015-12-31 | 2017-07-07 | 北京大学 | Influenza virus, its live vaccine of rite-directed mutagenesis and its preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| ADAM B. MILSTEIN等: ""Statistical approach for detection and localization of a fluorescing mouse tumor in Intralipid"", 《APPLIED OPTICS》 * |
| K. SCHNEIDER等，: ""Photoaffinity labelling with fluorescence detection"", 《HISTOCHEMISTRY》 * |
| KAORI SAKURAI等，: ""Photoaffinity labeling studies of the carbohydrate-binding proteins with different affinities"", 《BIOORG. MED. CHEM.》 * |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020024302A1 (en) * | 2018-08-03 | 2020-02-06 | 苏州大学张家港工业技术研究院 | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application therefor |
| CN110407873A (en) * | 2019-07-30 | 2019-11-05 | 苏州大学 | A kind of tumor microenvironment H2O2Respond cross-linking type near-infrared molecular probe and its application |
| CN110407873B (en) * | 2019-07-30 | 2020-10-16 | 苏州大学 | Tumor microenvironment H2O2Response crosslinking near-infrared molecular probe and application thereof |
| WO2021018099A1 (en) * | 2019-07-30 | 2021-02-04 | 苏州大学 | H2o2-responsive crosslinking near-infrared molecular probe for tumor microenvironment and use therefor |
| CN112521373B (en) * | 2019-09-19 | 2022-02-11 | 南京大学 | Multi-modal probe and preparation method and application thereof |
| CN112521373A (en) * | 2019-09-19 | 2021-03-19 | 南京大学 | Multi-modal probe and preparation method and application thereof |
| CN111803700A (en) * | 2020-07-15 | 2020-10-23 | 广东省医疗器械研究所 | Adhesive and preparation method thereof |
| WO2022099565A1 (en) * | 2020-11-12 | 2022-05-19 | 苏州大学 | Red-light-mediated nucleic acid anchoring-type fluorescent probe, and preparation method therefor and use thereof |
| CN112457682A (en) * | 2020-11-24 | 2021-03-09 | 浙江理工大学上虞工业技术研究院有限公司 | Disperse dye ink for digital ink-jet printing and preparation method and application thereof |
| CN112409815A (en) * | 2020-11-24 | 2021-02-26 | 浙江理工大学上虞工业技术研究院有限公司 | Bis-aziridine type disperse dyes having double reactive groups, process for their preparation and dyeing applications |
| CN112341838A (en) * | 2020-11-24 | 2021-02-09 | 浙江理工大学上虞工业技术研究院有限公司 | Reactive dye, reactive dye ink, and preparation method and application thereof |
| CN112239604B (en) * | 2020-11-24 | 2022-03-18 | 浙江理工大学 | Reactive disperse dye, preparation method and dyeing application thereof |
| CN112457682B (en) * | 2020-11-24 | 2022-04-08 | 浙江理工大学上虞工业技术研究院有限公司 | Disperse dye ink for digital ink-jet printing and preparation method and application thereof |
| CN112409815B (en) * | 2020-11-24 | 2022-04-08 | 浙江理工大学上虞工业技术研究院有限公司 | Bis-aziridine type disperse dyes having double reactive groups, process for their preparation and dyeing applications |
| CN112341838B (en) * | 2020-11-24 | 2022-05-10 | 浙江理工大学上虞工业技术研究院有限公司 | Reactive dye, reactive dye ink, and preparation method and application thereof |
| CN112239604A (en) * | 2020-11-24 | 2021-01-19 | 浙江理工大学 | Reactive disperse dye, preparation method and dyeing application thereof |
| CN114149482A (en) * | 2021-12-01 | 2022-03-08 | 苏州大学 | Intelligent conversion dual-stimulation response type probe for chelating metal ions as well as preparation method and application of probe |
| CN114149482B (en) * | 2021-12-01 | 2024-01-30 | 苏州大学 | Intelligent conversion dual-stimulus response type probe for chelating metal ions and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109180680B (en) | 2020-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109180680A (en) | A kind of ultraviolet light triggering cross-linking type near-infrared molecular probe and the preparation method and application thereof | |
| Patel et al. | Highly effective dual-function near-infrared (NIR) photosensitizer for fluorescence imaging and photodynamic therapy (PDT) of cancer | |
| CN106929003B (en) | A kind of multi-functional near infrared fluorescent probe and its preparation method and application | |
| CN107057398B (en) | A kind of application of seven methines cyanine dye and its accurate diagnosing and treating of tumour | |
| JP5823413B2 (en) | Process for the preparation of novel porphyrin derivatives and their use as PDT agents and fluorescent probes | |
| WO2004038378A2 (en) | Phthalocyanine dyes | |
| CN106866721B (en) | A kind of silicon phthalocyanine derivative and its application for preparing biotin acceptor targeting silicon phthalocyanine photosensitizer | |
| CN106008525B (en) | Micromolecule organic nano tumor photothermal treatment reagent and preparation method thereof | |
| CN109791107A (en) | Target the NIR dyestuff and application thereof of CA IX | |
| CN109096170A (en) | Nir dye, its targeted imaging agent, nano-carrier and anticancer drug and application | |
| CN109010826A (en) | A kind of targeting material based on Benzpyrole squaric acid cyanine dye and preparation method thereof and fluorescent nano particles and preparation method thereof | |
| EP3634397A1 (en) | Heterocyclyl polymethine ir chromophores | |
| CN113683602B (en) | Heptamethine cyanine micromolecule for multi-modal treatment of hypoxic tumors, and preparation method and application thereof | |
| CN112592386B (en) | Red light mediated nucleic acid anchoring type fluorescent probe and preparation method and application thereof | |
| US20220257798A1 (en) | H2o2-responsive crosslinking near-infrared molecular probe for tumor microenvironment and use therefor | |
| WO2023245857A1 (en) | Capsaicin-derived photosensitizer, and preparation method therefor and use thereof | |
| Patel et al. | Impact of Substituents in Tumor Uptake and Fluorescence Imaging Ability of Near‐Infrared Cyanine‐like Dyes | |
| CN106753337A (en) | A kind of near-infrared and two-photon Double-mode imaging fluorescence probe and its preparation and application | |
| CN108715591A (en) | Near infrared absorption porphyrin compound as photosensitizer and its application | |
| CN105198934B (en) | The platinum-like compounds of Photodynamic activity near infrared absorption, preparation method and applications | |
| CN109678888B (en) | Oxazine compound and application thereof | |
| WO2020024302A1 (en) | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application therefor | |
| CN113980013B (en) | Compound based on cyanine dye and preparation method and application thereof | |
| RU2548675C2 (en) | Amino amides in bacteriochlorophyll alpha series, having photodynamic activity and method for production thereof | |
| CN115337409B (en) | Albumin combined near infrared fluorescent probe-fatty acid conjugate, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200508 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |