CN109180680B - Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application thereof - Google Patents
Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN109180680B CN109180680B CN201810867119.7A CN201810867119A CN109180680B CN 109180680 B CN109180680 B CN 109180680B CN 201810867119 A CN201810867119 A CN 201810867119A CN 109180680 B CN109180680 B CN 109180680B
- Authority
- CN
- China
- Prior art keywords
- compound
- probe
- molecular probe
- infrared
- ultraviolet light
- 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.)
- Expired - Fee Related
Links
- 239000003068 molecular probe Substances 0.000 title claims abstract description 61
- 238000004132 cross linking Methods 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 230000001960 triggered effect Effects 0.000 title claims description 21
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 62
- 239000000523 sample Substances 0.000 claims abstract description 62
- 230000014759 maintenance of location Effects 0.000 claims abstract description 13
- 230000002035 prolonged effect Effects 0.000 claims abstract description 8
- 238000003745 diagnosis Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 37
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- 150000001875 compounds Chemical class 0.000 claims description 18
- 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 17
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 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 16
- 229940125898 compound 5 Drugs 0.000 claims description 15
- 229940125904 compound 1 Drugs 0.000 claims description 14
- 229940126214 compound 3 Drugs 0.000 claims description 14
- 229940125782 compound 2 Drugs 0.000 claims description 13
- 125000006239 protecting group Chemical group 0.000 claims description 13
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 12
- 235000019152 folic acid Nutrition 0.000 claims description 9
- 239000011724 folic acid Substances 0.000 claims description 9
- FZJACAROWOAKHE-JOCHJYFZSA-N (2R)-6-(9H-fluoren-1-ylmethoxycarbonylamino)-2-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C(C)(C)(C)OC(=O)N[C@H](CCCCNC(=O)OCC1=CC=CC=2C3=CC=CC=C3CC1=2)C(=O)O FZJACAROWOAKHE-JOCHJYFZSA-N 0.000 claims description 8
- 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 8
- 150000001408 amides Chemical class 0.000 claims description 8
- 238000006482 condensation reaction Methods 0.000 claims description 8
- 229960000304 folic acid Drugs 0.000 claims description 8
- JKANAVGODYYCQF-UHFFFAOYSA-N prop-2-yn-1-amine Chemical compound NCC#C JKANAVGODYYCQF-UHFFFAOYSA-N 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 6
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 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
- 150000002224 folic acids Chemical class 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000010511 deprotection reaction Methods 0.000 claims description 3
- 150000001540 azides Chemical class 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 31
- 210000004881 tumor cell Anatomy 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 10
- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical compound [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 abstract description 6
- 230000010354 integration Effects 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 239000000975 dye Substances 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000003384 imaging method Methods 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000011580 nude mouse model Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 241000699660 Mus musculus Species 0.000 description 6
- 238000000799 fluorescence microscopy Methods 0.000 description 6
- 230000001678 irradiating effect Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 238000007626 photothermal therapy Methods 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
- 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
- 238000000862 absorption spectrum Methods 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000011734 sodium Substances 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
- YXHKONLOYHBTNS-UHFFFAOYSA-N Diazomethane Chemical compound C=[N+]=[N-] YXHKONLOYHBTNS-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
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000004896 high resolution mass spectrometry Methods 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 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
- 229960004657 indocyanine green Drugs 0.000 description 3
- 238000006713 insertion reaction Methods 0.000 description 3
- 238000002953 preparative HPLC Methods 0.000 description 3
- 238000002390 rotary evaporation Methods 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 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
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 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
- 241001465754 Metazoa Species 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 230000017531 blood circulation Effects 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
- 230000003833 cell viability Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000144 sodium(I) superoxide Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
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)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (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 photo-crosslinking near-infrared molecular probe and a preparation method and application thereof. Specifically, the preparation method comprises the following steps: constructing and synthesizing a photo-crosslinking near-infrared molecular probe DACF; the photo-crosslinking near-infrared molecular probe DACF is crosslinked in cells under the trigger of ultraviolet light. The probe disclosed by the invention is irradiated by light with a specific wavelength to generate a high-activity carbene intermediate which is combined with a covalent bond of a biomolecule in a cell, so that the uptake of the probe in the cell is remarkably increased, the retention time of the probe is prolonged, and the long-time tracking of tumor cells can be realized. Meanwhile, the photo-thermal effect of the probe can be utilized to better kill tumor cells. The photo-crosslinking near-infrared molecular probe obtained by the preparation method can effectively improve the enrichment amount and retention time of the probe in the living tumor, realizes the diagnosis and treatment integration of the tumor, and has important scientific research and economic values.
Description
Technical Field
The invention belongs to the technical field of near-infrared dye functionalized modification, and particularly relates to a preparation method of ultraviolet-mediated bisaziridine group for traditional near-infrared dye Cypate modification, a molecular probe prepared by the method, and application of the probe in preparation of near-infrared fluorescence imaging of tumors and anti-tumor drugs based on photothermal therapy (PTT).
Background
As is well known, the organic near-infrared fluorescent dye has the characteristics of good fluorescence quantum yield, lower biotoxicity, plastic molecular structure, low price and the like, and is widely applied to near-infrared biological imaging.
Currently, the most common near-infrared dye approved by FDA for tumor imaging is Indocyanine Green (ICG), which can generate near-infrared fluorescence for near-infrared fluorescence imaging, and can also convert absorbed light energy into heat, raise local temperature, kill tumor cells, i.e., photothermal therapy, thereby realizing integration of tumor diagnosis and therapy. Although a large number of documents report that near-infrared fluorescent small-molecule probes synthesized based on the dyes can effectively target tumors and are used for tumor imaging and photothermal therapy, the dyes are easily eliminated by cell or tissue metabolism, so that the blood circulation period in vivo is short, and the imaging and photothermal therapy effects on the tumors are seriously influenced.
Therefore, the development of a new strategy and a new method capable of promoting the probe to be enriched at the tumor part and prolonging the retention time has great significance for improving the tumor imaging and treatment effects.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention constructs a photo-crosslinking near-infrared molecular probe, utilizes light to trigger crosslinking reaction, improves the enrichment amount of the probe in the tumor and prolongs the detention time of the probe, thereby effectively improving the imaging and treatment effects of the tumor; the method is suitable for various near-infrared dyes, and provides a new strategy and means for improving the long-time tracking and treatment effect of the near-infrared molecular probe on the tumor.
The invention adopts the following technical scheme:
an ultraviolet light triggered crosslinking near-infrared molecular probe has the following chemical structural formula:
the ultraviolet light triggered cross-linked near-infrared molecular probe is applied to the preparation of tumor diagnosis and/or treatment reagents.
The preparation method of the ultraviolet light triggered crosslinking near-infrared molecular probe comprises the following steps:
(1) carrying out amide condensation reaction on N-tert-butyloxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine and propargylamine to obtain a compound 1;
(2) removing a protecting group from the compound 1 to obtain a compound 2;
(3) reaction of compound 2 with NHS-activated bis-aziridine to give compound 3;
(4) removing a protecting group from the compound 3 to obtain a compound 4;
(5) reacting the compound 4 with a near-infrared dye to obtain a compound 5;
(6) and reacting the compound 5 with folic acid to obtain the ultraviolet light triggered crosslinking near-infrared molecular probe.
A preparation method of an acid-sensitive near-infrared dye comprises the following steps:
(1) carrying out amide condensation reaction on N-tert-butyloxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine and propargylamine to obtain a compound 1;
(2) removing a protecting group from the compound 1 to obtain a compound 2;
(3) reaction of compound 2 with NHS-activated bis-aziridine to give compound 3;
(4) removing a protecting group from the compound 3 to obtain a compound 4;
(5) and reacting the compound 4 with the near-infrared dye to obtain the acid-sensitive near-infrared dye.
A method for prolonging the retention time of a near-infrared molecular probe in a tumor comprises the following steps:
(1) carrying out amide condensation reaction on N-tert-butyloxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine and propargylamine to obtain a compound 1;
(2) removing a protecting group from the compound 1 to obtain a compound 2;
(3) reaction of compound 2 with NHS-activated bis-aziridine to give compound 3;
(4) removing a protecting group from the compound 3 to obtain a compound 4;
(5) reacting the compound 4 with a near-infrared dye to obtain a compound 5;
(6) reacting the compound 5 with folic acid to obtain an ultraviolet light triggered crosslinking near-infrared molecular probe;
(7) after the ultraviolet light triggered cross-linked near-infrared molecular probe is enriched at a tumor part, the residence time of the near-infrared molecular probe in the tumor is prolonged by ultraviolet light irradiation.
In the technical scheme, the ultraviolet light triggered cross-linked near-infrared molecular probe is dissolved in PBS (phosphate buffer salt, pH = 7.2-7.4) injection (the concentration is 45 mu M), injected into a tumor-bearing body, and then the probe is waited to reach the maximum enrichment amount at a tumor part, and after the probe is irradiated by ultraviolet light, the retention time of the near-infrared molecular probe at the tumor part is prolonged.
In the technical scheme, in the step (1), the molar ratio of N-tert-butyloxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine to propargylamine is 1: 1; the amide condensation reaction is carried out in the presence of HOBt, HBTU and diisopropylethylamine; the amide condensation reaction is carried out at room temperature for 15-24 hours.
In the technical scheme, in the step (2), the removal of the protective group of the compound 1 is carried out in a dichloromethane/trifluoroacetic acid mixed solvent; the volume ratio of the dichloromethane to the trifluoroacetic acid is 4: 1.
In the technical scheme, in the step (3), the reaction is carried out in the presence of diisopropylethylamine; compound 2, NHS-activated bisaziridine, diisopropylethylamine were present in a molar ratio of 1: 2.
In the technical scheme, in the step (4), the deprotection of the protecting group of the compound 3 is carried out in a mixed solvent of N, N-dimethylformamide and piperidine; the volume ratio of the N, N-dimethylformamide to the piperidine is 4: 1.
In the technical scheme, in the step (5), the molar ratio of the compound 4 to the near-infrared dye is 1.2: 1; the near-infrared dye is indocyanine green carboxyl derivative Cypate.
In the above technical scheme, in the step (6), the folic acid is azide-modified folic acid; the reaction of compound 5 with folic acid is carried out in the presence of sodium ascorbate and copper sulfate; the mol ratio of the compound 5, the folic acid, the sodium ascorbate and the copper sulfate is 1: 0.1: 0.05.
In the above technical scheme, in the step (7), when the ultraviolet light is irradiated, the wavelength is 365-405 nm, and the irradiation intensity is 1-12W/cm2The irradiation time is 1-10 minutes; the enrichment time of the probe at the tumor part is 0.5-1 hour.
In the above technical scheme, the chemical structural formulas of compound 1, compound 2, compound 3, compound 4 and compound 5 are as follows:
the chemical structure of the NHS-activated bis-aziridine is as follows:
the chemical structural formula of the azide-modified folic acid is as follows:
specifically, the method provided by the invention comprises the following steps:
(1) constructing and synthesizing a photo-crosslinking near-infrared molecular probe:
according to the designed synthesis steps: propargylamine is first subjected to an amide condensation reaction with N-tert-butoxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine, followed by 20% trifluoroacetic acid (dichloromethane: trifluoroacetic acid = 4: 1, v/v); removing Boc protective group of intermediate compound; then reacting with diazirine activated by NHS, removing a protecting group Fmoc of the obtained intermediate compound by using 20% piperidine (N, N-dimethylformamide: piperidine = 4: 1, v/v), carrying out an amide condensation reaction with a near infrared dye Cypate, and further carrying out a click chemical reaction with folic acid under the catalysis of sodium ascorbate and copper sulfate to obtain a final probe DACF, wherein the structural formula is as follows:
(2) the photo-crosslinking effect of the photo-crosslinking near-infrared molecular probe DACF in cells after ultraviolet irradiation is as follows:
dissolving the photo-crosslinking near-infrared molecular probe obtained in the step (1) in a cell culture medium, adding the solution into a 4T1 cell culture dish (concentration: 1 mu M), and placing the cell culture dish into an incubator for 12 hours. After the incubation time of the probe and the 4T1 cells is over, washing the cells once by PBS, adding a fresh culture medium, and irradiating the cells under ultraviolet light with the wavelength of 365-405 nm at the irradiation intensity of 1-12W/cm2The irradiation time is 1-10 minutes, and the molecular probe can perform a cross-linking reaction with macromolecular compounds such as protein in the tumor cells, so that the residence time of the molecular probe in the tumor cells is prolonged, and the long-time tracking of the tumor cells is facilitated.
(3) The photo-crosslinking function of the photo-crosslinking near-infrared molecular probe DACF in the tumor after ultraviolet irradiation:
dissolving the photo-crosslinking near-infrared molecular probe obtained in the step (1) in a PBS injection (concentration: 45 mu M), and injecting the probe into a BALB/c/nu female nude mouse with two tumors (4T 1 breast cancer) in a tail vein injection mode. After the probe is enriched for 0.5-1 hour at the tumor part, the probe is placed under the ultraviolet light with the wavelength of 365-405 nm for irradiation, and the irradiation intensity is 1-12W/cm2The irradiation time is 1-10 minutes, and the molecular probe can perform a cross-linking reaction with macromolecular compounds such as protein in the tumor cells, so that the residence time of the molecular probe in the tumor cells is prolonged, and the long-time tracking diagnosis of tumor parts is facilitated.
In the invention, after the compound 5 reacts with folic acid, semi-preparative high performance liquid chromatography is used for separation and purification to obtain the ultraviolet light triggered cross-linked near-infrared molecular probe, and the product is bluish solid powder. Preferably, the high performance liquid chromatography separation method comprises the following steps: c18 column, 3.5 μm, 4.6X 100 mm; mobile phase: a is trifluoroacetic acid: water = 1: 1000, parts by weight; b is trifluoroacetic acid: acetonitrile = 1: 1000, parts by weight; flow rate: 1 mL/min; linear gradient elution procedure: 0 min, A: b = 95: 5; 12 min, A: b = 0: 100.
in the invention, ultraviolet light irradiated by the ultraviolet light is provided by the ultraviolet light emitting device, the ultraviolet light emitting device is a handheld ultraviolet lamp or an ultraviolet laser, and the ultraviolet light emitting device is preferably an ultraviolet laser.
The preparation method of the invention improves the enrichment amount of the probe in the tumor cell and prolongs the retention time of the probe by light triggering crosslinking reaction.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
(1) in the invention, the diazirine (diazirine) functionalized modified near-infrared dye is used for the first time, and the light triggering condition is mild and simple;
(2) the photocrosslinking near-infrared molecular probe is triggered by ultraviolet light to perform photocrosslinking reaction without being influenced by external environment;
(3) after the probe enters a tumor cell, the probe is quickly converted into a carbene active intermediate under the irradiation of ultraviolet light, and then is quickly subjected to insertion reaction with C-H, O-H, N-H and S-H to crosslink on surrounding macromolecular protein through covalent bonds, so that the crosslinking reaction is very efficient.
Drawings
FIG. 1 is a flow chart of the synthesis of the photo-crosslinkable near-infrared molecular probe DACF in example 1;
FIG. 2 is a schematic diagram showing the mechanism of action of the photo-crosslinkable near-infrared molecular probe DACF in example 2;
FIG. 3 is the high performance liquid chromatography purity characterization (a) and the high resolution mass spectrometry characterization (b) of the photo-crosslinkable near-infrared molecular probe DACF in example 3;
FIG. 4 shows the ultraviolet absorption spectrum and fluorescence spectrum of the photo-crosslinkable near-infrared molecular probe DACF in example 4 (a) and the toxicity of the probe (b);
FIG. 5 is a fluorescent photograph of 4T1 cells surviving after irradiation with 405nm laser light for various periods of time in example 5;
FIG. 6 is a fluorescent photograph (a) and a corresponding standard fluorescent intensity map (b) of 4T1 cells at different times after irradiation with 405nm laser light in example 6;
FIG. 7 is a photograph (a) of near infrared fluorescence and a corresponding graph (b) of standard fluorescence signal intensity of a tumor site of a mouse at different times after irradiation of 405nm laser in example 7;
FIG. 8 shows photoacoustic photographs (a) of mouse tumor sites and corresponding standard photoacoustic signal values (b) at different times after irradiation of 405nm laser in example 8.
Detailed Description
The invention will be further elucidated with reference to the drawings and specific embodiments. It should be understood that these examples are only for explaining and illustrating the technical solutions of the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise specified, materials, reagents, instruments and the like used in the following examples are commercially available.
Example 1: synthesis and characterization of photo-crosslinking near-infrared molecular probe
(1) A100 mL round bottom flask was charged with propargylamine (0.29 mL), N-dimethylformamide (30 mL), HOBt (0.68 g, 5.04 mmol), HBTU (1.91 g, 5.04 mmol) and diisopropylethylamine (1.08 g, 8.4 mmol) and stirred continuously. Subsequently, N-t-butyloxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine (1.97 g, 4.2 mmol) was added to the reaction flask, and stirring was continued and the reaction was allowed to proceed overnight (20 hours) at room temperature. After the reaction was complete, the solvent was removed by rotary evaporation, and 30 mL of ethyl acetate was added to redissolve the intermediate compound. The organic phase was then washed once with 30 mL each of deionized water, saturated sodium bicarbonate and aqueous sodium chloride, dried over anhydrous magnesium sulfate and then spin-dried to give intermediate 1 (the structure of which is shown as Compound 1 in FIG. 1) as a white powder (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 (d，J = 5.1 Hz，1H)，3.86 (dd，J = 5.2，2.3 Hz，2H)，3.10 (t，J = 2.5 Hz，1H)，2.89 (d，J = 2.7 Hz，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’dfor C29H35N3NaO5[M+Na]+，528.2474；found，528.2470。
(2) A50 mL round bottom flask was charged with 16 mL of methylene chloride and 4 mL of trifluoroacetic acid and stirred well. Intermediate compound 1 (1.01 g, 2 mmol) was then added to the reaction flask and the reaction stirred at room temperature for 1 h. After completion of the reaction, the reaction solution was removed by rotary evaporation and purified by silica gel column chromatography (dichloromethane: methanol = 10: 1, v/v) to obtain intermediate 2 (the structure is shown as compound 2 in fig. 1) as a white solid (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 (t，J = 7.3 Hz，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) In a 50 mL round bottom flask were added 15 mL of dichloromethane, NHS activated bisazirine (NHS-diazirine) (0.25 g, 1 mmol), intermediate compound 2 (0.41 g, 1 mmol) and diisopropylethylamine (278. mu.L, 2 mmol), and the reaction was stirred at room temperature for 2 h. After the reaction was completed, the organic phase was washed three times with 25 mL of deionized water, once with 25 mL of sodium chloride aqueous solution, dried over anhydrous magnesium sulfate and then spin-dried. Subsequent purification by silica gel column chromatography (dichloromethane: methanol = 20: 1, v/v) gave intermediate 3 (the structure of which is shown as compound 3 in fig. 1) as a white solid (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 (t，J = 7.4 Hz，2H)，7.30 (t，J = 7.4 Hz，2H)，4.21 (dt，J = 16.2，6.8 Hz，3H)，3.90 (dd，J = 13.7，8.8Hz，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 = 39.2Hz，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) A50 mL round bottom flask was charged with 8 mL of N, N-dimethylformamide and 2 mL of piperidine and stirred well. Intermediate compound 3 (0.5 g, 0.97 mmol) was then added to the reaction flask and the reaction stirred at room temperature for 1 h. After completion of the reaction, the reaction mixture was removed by rotary evaporation and purified by silica gel column chromatography (dichloromethane: methanol = 10: 1, v/v) to obtain intermediate 4 (the structure is shown as compound 4 in fig. 1) as a yellow oil (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 = 5.8Hz，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 forC14H24N5NaO2[M+Na]+，316.1749；found，316.1737。
(5) In a 20 mL round bottom flask were added HOBt (4.7 mg, 0.035 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (6.7 mg, 0.035 mmol) and Cypate (18.1 mg, 0.029 mmol), and activated with stirring at room temperature for 20 min. Intermediate compound 4 (10.3 mg, 0.035 mmol) was then added to the reaction flask and the reaction was stirred at room temperature for an additional 8 h. After the completion of the reaction, the reaction mixture was purified by semi-preparative high performance liquid chromatography to give intermediate 5 (whose structure is shown as Compound 5 in FIG. 1) as a bluish solid powder (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 = 3.6Hz，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), intermediate compound 5 (9 mg, 0.01 mmol) and the folate modified azide molecule (5.2 mg, 0.01 mmol) were added to a 5 mL round bottom flask and stirred well. Meanwhile, sodium ascorbate (0.2 mg, 10 mmol%) and anhydrous copper sulfate (0.09 mg, 5 mmol%) were mixed and dissolved in 1 mL of deionized water, and the mixture was added to the reaction flask and stirred at room temperature for reaction for 8 h. After the completion of the reaction, it was separated and purified by semi-preparative high performance liquid chromatography to obtain molecular probe DACF (bluish solid powder, 12.8 mg, yield: 94%).
HRMS (ESI)：m/z calcd for C77H87N18O10 +[M]+，1423.6847；found，1423.1622。
Example 2: photocrosslinking effect of DACF (DACF) of photocrosslinking near-infrared molecular probe after ultraviolet irradiation
As shown in FIG. 2, the photo-crosslinkable near-infrared molecular probe DACF prepared in example 1 was diluted in a cell culture medium, and then added to a 4T1 cell culture dish for incubation. After the probe enters a tumor cell, a group carried by the probe generates intermediate carbene through ultraviolet irradiation, and then the intermediate carbene is combined with surrounding macromolecular proteins and other covalent bonds through C-H, O-H, N-H and S-H insertion reaction, so that the probe molecules are firmly connected with the macromolecular proteins and the like in the tumor cell, the retention time of the probe molecules in the cell is prolonged, and the imaging and treatment effects of the probe molecules are improved, wherein the mechanism is shown in figure 2.
Example 3: high performance liquid chromatography purity characterization and high resolution mass spectrometry characterization of light-crosslinked near-infrared molecular probe DACF
The photo-crosslinking near-infrared molecular probe prepared in example 1 was diluted with a solvent methanol to a concentration of 5 μ M, and then molecular weight determination was performed on the probe by high-resolution mass spectrometry, and purity analysis was performed thereon using a high performance liquid chromatograph.
As shown in fig. 3a, the sample was analyzed by Agilent 1260 hplc, the retention time of the probe DACF was 8.176 minutes, the peak areas were further integrated, and the concentration of the probe in the sample was calculated to be as high as 99%. FIG. 3b shows the theoretical m/z of probe DACF: 1423.6847, actually obtaining m/z in a high-resolution mass spectrum: 1423.1622, the two are identical, thus obtaining the desired compound.
Example 4: ultraviolet absorption spectrum and fluorescence spectrum conditions of DACF (fluorescent Desorption fluorescence) of photo-crosslinking near-infrared molecular probe and toxicity condition of probe
Diluting the photo-crosslinking near-infrared molecular probe prepared in the embodiment 1 to the concentration of 5 mu M by using a solvent, and measuring an ultraviolet absorption spectrum and a fluorescence spectrum of the probe by using an ultraviolet-visible absorption spectrometer and a steady-state/transient-state fluorescence spectrometer respectively; the cytotoxicity of the probe against 4T1 was examined by MTT (thiazole blue) colorimetry.
As shown in FIG. 4a, the probe DACF has two distinct absorption peaks in the range of 650-800 nm as shown by the ultraviolet absorption spectrum, and the maximum absorption peak is 785 nm; the probe DACF has an emission peak in the range of 805-900 nm and the maximum emission is 830 nm as shown by fluorescence spectrum.
FIG. 4b shows the cytotoxicity test of molecular probe DACF, the survival rate of cells is dependent on the concentration of the probe, and the toxicity of the probe to the cells is increased along with the increase of the concentration. However, the molecular probe DACF still has a cell viability of more than 80% at a concentration of 14. mu.M, which is far higher than the concentration of the probe used in clinical practice.
Example 5: damage to 4T1 cells by 405nm laser irradiated at different times
Cytotoxicity test methods: mouse breast cancer cells (4T 1) in 6-well plates (density 3X 10)5One/hole), incubating for 24 h, and irradiating with ultraviolet laser (405 nm, power 1W/cm)2) Irradiating for different time (0, 1, 2, 4, 8, 10 min), placing into an incubator, continuously culturing for 4 h, removing supernatant, adding Live-Dead staining solution, culturing in the incubator for 30 min, and photographing with a Leica fluorescence microscope after staining.
As shown in fig. 5, when the time for irradiating the 405nm laser was within 4 minutes, the survival rate of the 4T1 cells was remarkably high, and there were substantially no dead cells, as compared with the control group not irradiated with the laser. However, when the irradiation time of the 405nm laser is 8 minutes or more, the 4T1 cells are strongly damaged, and cell death occurs in a large area. Therefore, the time for irradiating the laser light of 405nm is preferably 1.5 minutes.
Example 6: the retention condition of the photo-crosslinking near-infrared molecular probe DACF in 4T1 cells after ultraviolet irradiation
4T1 cells were seeded in confocal dishes of 8-well plates, 5X 10 cells per well, based on the method described in example 23The cells were cultured in an incubator for 24 hours. Then the medium was discarded, the same concentration of cell culture medium containing the probe (probe concentration 1. mu.M), 200. mu.L were added, the mixture was cultured in an incubator for 12 hours, and after the incubation time of the probe with 4T1 cells was completed, a 405nm laser (1W/cm)2) Irradiating for 1.5 min while setting experiment control group, adding fresh culture medium, and culturing in incubator for 8 hr, 24 hr and 48 hr. Then staining the nucleus with Hoechst 33342 staining solution, and observing the fluorescence condition in 4T1 cells in different time after the irradiation of 405nm laser by using a laser confocal microscope
As shown in FIG. 6a, the intensity of red fluorescence was the same in 4T1 cells at the initial 0 h. At 8 h after the irradiation of the 405nm laser, the red fluorescence in the 4T1 cells was significantly stronger than that of the control group which was not irradiated with the 405nm laser. And the fluorescence of the control group not irradiated with the 405nm laser decreased very rapidly with the lapse of time, and after 24 h, almost no red fluorescence signal was observed in the 4T1 cells. Compared with the experimental group irradiated with the laser at 405nm, obvious red fluorescence can be observed in the 4T1 cells at 48 h. FIG. 6b is the corresponding fluorescence quantitative data, and the experimental group irradiated with 405nm laser can prolong the retention time of the probe in the cell for 48 h, while the control group not irradiated with 405nm laser can only retain the retention time in the cell for 24 h. Therefore, the light-crosslinking near-infrared molecular probe DACF can be potentially applied to cell tracing.
Example 7: near-infrared fluorescence imaging condition of photo-crosslinking near-infrared molecular probe DACF at mouse tumor part
The probe DACF was injected into BALB/c/nu female nude mice bearing two tumors by tail vein injection at a dose of 45. mu.M (200. mu.L/each) based on the method described in example 2. Half an hour after injection of the probe, a 405nm laser (1W/cm) was used2) The left tumor was irradiated with laser for 10 min, while the right tumor was not irradiated with 405nm laser as a control group. Then put in a small motionIn the IVIS Lumina XRMS in-vivo imaging system, the distribution of the probes in two tumors of the nude mice is observed, and the fluorescence intensity of the two tumor parts of the nude mice at different time points is calculated through IVIS in-vivo imaging analysis software.
As shown in FIG. 7a, at 1 h, the fluorescence signal intensity of the molecular probe was consistent in the left and right tumors of the mouse. Then, the probe was metabolized in the mouse with time, and at 8 h, the fluorescence signal of the tumor part not irradiated with the 405nm laser on the right side was gradually reduced and was lower than that of the tumor part irradiated with the 405nm laser on the left side. At 12h, the fluorescence signal of the tumor part which is not irradiated with the 405nm laser on the right side is almost zero, and the fluorescence signal of the tumor part which is irradiated with the 405nm laser on the left side is still obvious up to 24 h. From the fluorescence signal statistics of the tumor sites in FIG. 7b, it can be clearly seen that the tumor sites without 405nm laser irradiation have no fluorescence intensity signal at 12h, while the experiment group tumor sites have a fluorescence signal lasting for 24 h. The result of in vivo fluorescence imaging proves that the photocrosslinking probe can carry out photocrosslinking reaction at a tumor part, is crosslinked in a tumor, reduces cell metabolism efflux, and enables the probe to be retained at the tumor part for a long time, thereby prolonging the fluorescence imaging time.
Example 8: photoacoustic imaging condition of photo-crosslinking near-infrared molecular probe DACF at mouse tumor site
BALB/c/nu female nude mice bearing two tumors were injected with DACF probe by tail vein injection at 45 μ M (200 μ L/each). Half an hour after injection of the probe, a 405nm laser (1W/cm) was used2) The left tumor was irradiated with laser for 10 min, while the right tumor was not irradiated with 405nm laser as a control group. Simultaneously, the small animal photoacoustic imaging system is opened, and the water temperature in the water bath of the photoacoustic imager reaches 37 DEGoAnd C, placing the anesthetized nude mice, and scanning two tumor part images of the nude mice. The obtained photoacoustic imaging data was then subjected to reconstruction analysis using MSOT inSight/inVision analysis software.
As can be seen from fig. 8a, at 8 h, the tumor site irradiated with 405nm laser exhibited a strong photoacoustic signal, the tumor site not irradiated with 405nm laser had already a weak photoacoustic signal, and at 12h, the photoacoustic signal had nearly disappeared. Then the tumor site irradiated with 405nm laser still shows clear photoacoustic signals at 24 h. From the photoacoustic signal statistics of the tumor site of fig. 8b, it can also be observed that the tumor site irradiated with the 405nm laser still has a strong photoacoustic signal until 24 h, while the control group photoacoustic signal value has approached 0. By combining the results of the fluorescence imaging and the photoacoustic imaging, the DACF probe can perform a photocrosslinking reaction at a tumor part, is crosslinked in a tumor, reduces the discharge of cell metabolism, and enables the probe to be retained at the tumor part for a long time, thereby prolonging the photoacoustic imaging time.
The application of photosensitive probes to photocrosslinkable labeled proteins has been studied and developed. In order to overcome the defects of the traditional near-infrared molecular probe, the photo-crosslinking near-infrared molecular probe is constructed, and the crosslinking reaction is triggered by light, so that the enrichment amount of the probe on a tumor part is increased, the retention time of the probe is prolonged, and the imaging effect of the tumor is effectively improved. It has the following advantages: firstly, the light triggering condition is mild and simple; secondly, the photo-crosslinking reaction is not influenced by conditions such as the pH value of a tumor part; thirdly, after the probe enters the tumor cell, the probe is quickly converted into a carbene active intermediate under the irradiation of ultraviolet light, and then the carbene active intermediate is quickly subjected to insertion reaction with C-H, O-H, N-H and S-H to be crosslinked on surrounding macromolecular protein through covalent bonds, so that the crosslinking reaction is very efficient. Therefore, the near-infrared molecular probe can be further applied to the fields of biology and the like.
Claims (7)
1. The ultraviolet light triggered crosslinking near-infrared molecular probe is characterized by having the following chemical structural formula:
2. the ultraviolet light-triggered cross-linked near-infrared molecular probe of claim 1, wherein the preparation method of the ultraviolet light-triggered cross-linked near-infrared molecular probe comprises the following steps:
(1) carrying out amide condensation reaction on N-tert-butyloxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine and propargylamine to obtain a compound 1;
(2) removing a protecting group from the compound 1 to obtain a compound 2;
(3) reaction of compound 2 with NHS-activated bis-aziridine to give compound 3;
(4) removing a protecting group from the compound 3 to obtain a compound 4;
(5) reacting the compound 4 with a near-infrared dye to obtain a compound 5;
(6) reacting the compound 5 with azide-modified folic acid to obtain the ultraviolet light-triggered cross-linked near-infrared molecular probe;
the chemical structural formulas of the compound 1, the compound 2, the compound 3, the compound 4 and the compound 5 are respectively as follows:
the chemical structure of the NHS-activated bis-aziridine is as follows:
the chemical structural formula of the azide-modified folic acid is as follows:
3. the ultraviolet-triggered cross-linking near-infrared molecular probe of claim 2, wherein the molar ratio of N-tert-butoxycarbonyl-N' -fluorenylmethoxycarbonyl-D-lysine to propargylamine is 1: 1; the deprotection of the protecting group of the compound 1 is carried out in a mixed solvent of dichloromethane and trifluoroacetic acid.
4. The ultraviolet light-triggered cross-linking near-infrared molecular probe according to claim 2, wherein the deprotection of the protecting group of the compound 3 is carried out in a mixed solvent of N, N-dimethylformamide and piperidine; the molar ratio of the compound 4 to the near-infrared dye is 1.2: 1.
5. The ultraviolet-triggered cross-linked near-infrared molecular probe as claimed in claim 2, wherein the reaction of compound 5 with the azide-modified folic acid is carried out in the presence of sodium ascorbate and copper sulfate; the mol ratio of the compound 5, the folic acid modified by the azide, the sodium ascorbate and the copper sulfate is 1: 0.1: 0.05.
6. Use of the UV-triggered cross-linked NIR molecular probe of claim 1 for preparing a probe with prolonged retention time in tumors.
7. The use of the ultraviolet light-triggered cross-linked near-infrared molecular probe of claim 1 in the preparation of a tumor diagnosis and/or treatment reagent.
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 CN109180680A (en) | 2019-01-11 |
| CN109180680B true 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) |
Families Citing this family (10)
| 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 |
| CN110407873B (en) * | 2019-07-30 | 2020-10-16 | 苏州大学 | Tumor microenvironment H2O2Response crosslinking near-infrared molecular probe and application thereof |
| CN112521373B (en) * | 2019-09-19 | 2022-02-11 | 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 |
| US20230407169A1 (en) * | 2020-11-12 | 2023-12-21 | Soochow University | Red-light-mediated nucleic acid anchoring-type fluorescent probe, and preparation method therefor and use thereof |
| 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 |
| 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 |
| 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 |
| CN112239604B (en) * | 2020-11-24 | 2022-03-18 | 浙江理工大学 | Reactive disperse dye, preparation method and dyeing application thereof |
| 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 |
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 |
|---|
| "Photoaffinity labeling studies of the carbohydrate-binding proteins with different affinities";Kaori Sakurai等，;《Bioorg. Med. Chem.》;20150731;第23卷;第5319–5325页 * |
| "Photoaffinity labelling with fluorescence detection";K. Schneider等，;《Histochemistry》;19941231;第101卷;第135-142页 * |
| "Statistical approach for detection and localization of a fluorescing mouse tumor in Intralipid";Adam B. Milstein等;《Applied Optics》;20050420;第44卷(第12期);第2300-2310页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109180680A (en) | 2019-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109180680B (en) | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application thereof | |
| CN103003282B (en) | A kind of prepare new derivatives of porphyrin method and purposes as PDT reagent and fluorescent probe | |
| US20040171827A1 (en) | Phthalocyanine dyes | |
| CN109320536B (en) | Aza-BODIPY based near-infrared two-window fluorescent probe and preparation and application thereof | |
| Lv et al. | Synthesis, properties and near-infrared imaging evaluation of glucose conjugated zinc phthalocyanine via Click reaction | |
| CN110003461B (en) | Polyiodide-modified BODIPY derivative and preparation method and application thereof | |
| CN106929003A (en) | A kind of multi-functional near infrared fluorescent probe and its preparation method and application | |
| WO2023001317A1 (en) | Acid-responsive nano-micelle on basis of cherenkov effect, and preparation method and use therefor | |
| CN113683602B (en) | Heptamethine cyanine micromolecule for multi-modal treatment of hypoxic tumors, and preparation method and application thereof | |
| Lv et al. | A novel water-soluble near-infrared glucose-conjugated porphyrin: synthesis, properties and its optical imaging effect | |
| CN112592386A (en) | Red light mediated nucleic acid anchoring type fluorescent probe and preparation method and application thereof | |
| CN110407873B (en) | Tumor microenvironment H2O2Response crosslinking near-infrared molecular probe and application thereof | |
| Patel et al. | Impact of Substituents in Tumor Uptake and Fluorescence Imaging Ability of Near‐Infrared Cyanine‐like Dyes | |
| CN111606839A (en) | Diagnosis and treatment combined molecular lead compound synthesis method and application in mitochondrial imaging | |
| WO2023245857A1 (en) | Capsaicin-derived photosensitizer, and preparation method therefor and use thereof | |
| CN107522773B (en) | Pentapeptide modified rhodamine B compound and preparation method and application thereof | |
| CN106753337A (en) | A kind of near-infrared and two-photon Double-mode imaging fluorescence probe and its preparation and application | |
| KR101842633B1 (en) | Diagnostic or therapeutic composition of cancer which is activated by cathepsin B, and near-infrared imaging and phototherapy of tumor using the same | |
| CN116217515A (en) | Compound, preparation method thereof and application thereof in detection of cysteine | |
| WO2020024302A1 (en) | Ultraviolet light triggered crosslinking near-infrared molecular probe and preparation method and application therefor | |
| Wilson et al. | Tissue oxygen measurements using phosphorescence quenching | |
| RU2372099C1 (en) | Ytterbium tetrapyrazolyl porphyrin complexes as fluorescent tags to diagnose malignant growths | |
| CN114835629B (en) | Carbazole benzo [ cd ] indolium salt and preparation method and application thereof | |
| CN115028572B (en) | Carbazole benzo [ e ] indole hybrid compound, and preparation method and application 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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200508 |