CN109054013A - A kind of modified indocyanine green and preparation method thereof - Google Patents
A kind of modified indocyanine green and preparation method thereof Download PDFInfo
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- CN109054013A CN109054013A CN201810959509.7A CN201810959509A CN109054013A CN 109054013 A CN109054013 A CN 109054013A CN 201810959509 A CN201810959509 A CN 201810959509A CN 109054013 A CN109054013 A CN 109054013A
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- indocyanine green
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- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical class [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 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 27
- 238000012986 modification Methods 0.000 claims abstract description 20
- 229960004657 indocyanine green Drugs 0.000 claims abstract description 16
- 229920001223 polyethylene glycol Polymers 0.000 claims description 58
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 14
- 150000002148 esters Chemical group 0.000 claims description 6
- 239000000376 reactant Substances 0.000 claims description 6
- MSKSQCLPULZWNO-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanamine Chemical compound COCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCN MSKSQCLPULZWNO-UHFFFAOYSA-N 0.000 claims description 5
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 5
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 238000005034 decoration Methods 0.000 claims description 3
- 229920001427 mPEG Polymers 0.000 claims description 3
- ASUGWWOMVNVWAW-UHFFFAOYSA-N 1-(2-methoxyethyl)pyrrole-2,5-dione Chemical compound COCCN1C(=O)C=CC1=O ASUGWWOMVNVWAW-UHFFFAOYSA-N 0.000 claims description 2
- ASDQMECUMYIVBG-UHFFFAOYSA-N 2-[2-(2-aminoethoxy)ethoxy]ethanol Chemical compound NCCOCCOCCO ASDQMECUMYIVBG-UHFFFAOYSA-N 0.000 claims description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims 1
- 230000004048 modification Effects 0.000 abstract description 19
- 206010028980 Neoplasm Diseases 0.000 abstract description 17
- 210000004369 blood Anatomy 0.000 abstract description 13
- 239000008280 blood Substances 0.000 abstract description 13
- 238000001514 detection method Methods 0.000 abstract description 9
- 230000003595 spectral effect Effects 0.000 abstract description 8
- 238000002583 angiography Methods 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 description 25
- 241000699666 Mus <mouse, genus> Species 0.000 description 22
- 238000002347 injection Methods 0.000 description 22
- 239000007924 injection Substances 0.000 description 22
- 239000002202 Polyethylene glycol Substances 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 238000000799 fluorescence microscopy Methods 0.000 description 11
- 238000003384 imaging method Methods 0.000 description 10
- KDJUNNVUQBKNAY-UHFFFAOYSA-N 4-[(2E)-2-[(2E,4E,6E)-7-[3-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-1,1-dimethylbenzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonate Chemical compound CC1(C)\C(=C/C=C/C=C/C=C/C2=[N+](CCCCS([O-])(=O)=O)C3=C(C4=C(C=CC=C4)C=C3)C2(C)C)N(CCCCCC(=O)ON2C(=O)CCC2=O)C2=C1C1=C(C=CC=C1)C=C2 KDJUNNVUQBKNAY-UHFFFAOYSA-N 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 210000002381 plasma Anatomy 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 8
- 229920001400 block copolymer Polymers 0.000 description 8
- 210000004204 blood vessel Anatomy 0.000 description 8
- 210000003462 vein Anatomy 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 125000004185 ester group Chemical group 0.000 description 6
- 239000012894 fetal calf serum Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 4
- 230000017531 blood circulation Effects 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 229920001610 polycaprolactone Polymers 0.000 description 4
- -1 succinimide ester Chemical class 0.000 description 4
- 230000002792 vascular Effects 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 238000000295 emission spectrum Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 229920002239 polyacrylonitrile Polymers 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000003191 femoral vein Anatomy 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- OFXSXYCSPVKZPF-UHFFFAOYSA-N methoxyperoxymethane Chemical compound COOOC OFXSXYCSPVKZPF-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229920001195 polyisoprene Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- NBBJYMSMWIIQGU-UHFFFAOYSA-N propionic aldehyde Natural products CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 238000002601 radiography Methods 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 229960002317 succinimide Drugs 0.000 description 2
- 230000036326 tumor accumulation Effects 0.000 description 2
- 241000415078 Anemone hepatica Species 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 210000003445 biliary tract Anatomy 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000012661 block copolymerization Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000013534 fluorescein angiography Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002357 laparoscopic surgery Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 238000007626 photothermal therapy Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008337 systemic blood flow Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000001392 ultraviolet--visible--near infrared spectroscopy Methods 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/334—Polymers modified by chemical after-treatment with organic compounds containing sulfur
- C08G65/3348—Polymers modified by chemical after-treatment with organic compounds containing sulfur containing nitrogen in addition to sulfur
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G2650/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterized by the type of post-polymerisation functionalisation
- C08G2650/04—End-capping
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G2650/28—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type
- C08G2650/50—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule characterised by the polymer type containing nitrogen, e.g. polyetheramines or Jeffamines(r)
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to a kind of modified indocyanine green, being will be replaced indocyanine green molecular structure two sulfonic one of being modified property of sulfonic group PEG group.By carrying out modification to ICG, the plasma half-life of ICG can be extended, its residence time in blood is made to be significantly longer, so that navigation, the detection of tumour and various applications such as treatment are optimised in angiography, art.The ICG after modifying and original ICG show close good photoluminescent property and spectral absorptive capacity, and stability is preferable, have the prospect applied very well.The invention further relates to the preparation methods of the modified ICG.
Description
Technical field
The present invention relates to the method for modifying to Fluorescein angiography agent, especially with regard to the indocyanine green modified with PEG and
Preparation method.
Background technique
Indocyanine green (Indocyanine Green) is a kind of tricarbocyanine dye that molecular weight is 751.4Da.It is one
The ion of kind negative electrical charge, belongs to big cyanine dye family.Indocyanine green structural formula A referring to Figure 1 of the drawings, is that one kind is commonly used to examine
Look into the dyestuff medicine of liver function and the effective blood flow of liver.After being injected intravenously in vivo, combined at once with plasma protein, it is fast with blood circulation
Speed is distributed in system vascular, and high efficiency is selectively absorbed by liver cell, and is excreted to bile in a free form from liver cell
In, enter intestines through biliary tract, is excreted with excrement.It is fast due to draining, general normal person's intravenous injection after twenty minutes there are about 97% from
Excluded in blood, be not involved in vivo chemical reaction, without intestines liver circulation (into the ICG no longer absorbed into serum of intestinal tube), without lymph adverse current,
Not outside other livers such as kidney, internal organs are drained.
The angiographic applications of ICG during surgery are extensive, with other angiographic method (X-ray, CT, MRI and PET) phases
Than, can easily and economically inject ICG carry out angiography, such as neurosurgery, the coronary artery surgery that detours,
In flap surgery, reconstruction operations, trauma operation and laparoscopic surgery, it is used to check in blood circulation and art and navigates.ICG is pushed away
Note enters systemic blood circulation, and completes imaging in a few minutes after injection, as needed usually can be after about 15 minutes again
Secondary injection.
ICG is also used for lesion detection and treatment.The fluorescence quantum yield of ICG about 10%, and fluorescence and photoacoustic signal
It is increased and decreased respectively with fluorescence quantum yield, when an ICG molecule is excited, it can be further by energy transmission
Give other molecules.So ICG may be used as the contrast agent of fluorescence imaging and photoacoustic imaging.ICG can pass through EPR effect, non-spy
The opposite sex is in tumor accumulation or targeting modification, and specificity is in tumor accumulation, to carry out the detection of tumour, positioning and progress
Photo-thermal therapy and photodynamic therapy.
But since half-life period of the ICG in blood plasma is very short, limit ICG answering as near-infrared fluorescence imaging material
With, and it is clinical be all at present according to using multiple injection or slowly injecting by the way of, for the clinical accurate fluorescence detection of acquisition
As a result, bringing difficulty.For example, when measurement hepatic blood flow be dissolved in 25mgICG in sterile water for injection as a small amount of as possible,
The above-mentioned solution for being equivalent to 3mgICG is injected intravenously at 2.5~5.0mg/ml concentration with normal saline dilution again.Then, with every
Minute 0.27~0.49mg constant (while need to be adopted until having adopted blood sample with certain speed intravenous drip about 50 minutes
PeV and vena hepatica blood).
Summary of the invention
In order to solve the above-mentioned technical problem, present invention contemplates that by the way that existing ICG is modified or is modified, to extend
The plasma half-life of ICG saves the fiber crops needed in the test of Blood fluorescence radiography to sufferer multiple injection or slow continuous injection
It is tired.The ICG after modifying has the good photoluminescent property and spectral absorptive capacity close with former ICG, and stability simultaneously
Preferably, there is the prospect applied very well.
In order to achieve the above object, the main technical schemes that the present invention uses include:
A kind of modified indocyanine green, being will be replaced being modified property of-NHS PEG group in indocyanine green molecular structure.
Wherein, the modified PEG is called modified polyethylene glycol, is by chemical modification group or bioactivity base
The PEG of group's modification.Modified PEG group refer to modified PEG lose end H atom after be formed by active group.
Modified PEG includes but is not limited to mPEG (mono methoxy ether PEG), mPEG-NH2 (mPEG- amido), mPEG-SS
(mPEG- succinimidyl succinate), mPEG-SC (mPEG-SC), mPEG2-NHS (mPEG2-N- hydroxyl
Base succinimide ester), mPEG-SPA (mPEG- succinimide propionic acid acid esters), mPEG-ALD (mPEG- propionic aldehyde), mPEG-
MAL (mPEG- maleimide), HO-PEG-COOH (a- hydroxyl-w- carboxy polyethylene glycol), mPEG-b-PS (polyethylene glycol and
The block copolymer of polystyrene), mPEG-b-PI (block copolymer of polyethylene glycol and polyisoprene), mPEG-b-PAN
(block copolymer of the polyethylene glycol and polyacrylonitrile), mPEG-b-PCL (block copolymerization of polyethylene glycol and poly-epsilon-caprolactone
Object), mPEG-b-PMMA (block copolymer of polyethylene glycol and polymethyl methacrylate), α-hydroxyl-PEG- ω-
Amide, double hydroxyl PEG, HO-PEG-NH2 (Alpha-hydroxy-omega-amino polyethylene glycol), mPEG-carboxyl (mPEG- carboxyl),
MPEG-cyan (mPEG- itrile group) etc..
A preferred embodiment according to the present invention, the molecular weight value range of the modified PEG group is 500~
20000, preferably 500,2000,5000,10000,20000.
The molecular structural formula of a preferred embodiment according to the present invention, the indocyanine green through PEG modification is expressed as follows:
Wherein, n=9~450.
According to the present invention, a kind of method for preparing modified indocyanine green, the indoles cyanines modified through PEG are further related to
Green molecular structural formula is expressed as follows:
Wherein n=9~450；The described method includes:
The ICG derivative and mPEG-NH modified with warp-CO-NHS ester group2For reactant, mPEG-NH2With the ICG derivative
In-CO-NHS occur ester substitution reaction, slough a molecule n-hydroxysuccinimide, be made modified by mPEG-NH- it is novel
ICG structural formula C；Its reaction process indicates are as follows:
Preferably, the reaction condition of above-mentioned reaction process are as follows: the ICG derivative of warp-CO-NHS ester group modification with
mPEG-NH21:5 is mixed in molar ratio, and in the PBS of pH=7.0~8.0, room temperature shaker reacts 5~12h, is purified through HPLC etc.
Processing, obtains PEG-ICG, structural formula C.
Main inventive of the invention is to be repaired indocyanine green molecular structure two sulfonic one of sulfonic groups
Replaced decorations property PEG group, for extending the plasma half-life of ICG, while ICG is not influenced even also to further strengthen in blood vessel
The application performance of radiography, blood vessel fluorescence imaging, EPR effect etc..But the sulfonic group of ICG, which is difficult to directly react, replaces with modification
Property PEG, thus the preparation method is that using warp-CO-NHS ester group modification ICG derivative as reactant rather than ICG[this
ICG derivative both can be synthesized voluntarily, also commercially available], it is since-CO-NHS ester group activity therein is stronger, it is easy to
mPEG-NH2Transesterification occurs, sloughs a molecule NHS, mPEG-NH- is connected on carbonyl-C=O-, is made therefrom through one
The novel I CG that kind is modified through PEG completes the process that ICG is modified by PEG.Wherein, if using different molecular weight mPEG-NH2For
Reactant then after reaction so that the mPEG-NH- of various different molecular weights is keyed on-C=O-, reaches different points of completion
The purpose of son amount PEG modification, reaction controllability is strong, can design reaction product as needed, and reaction condition is mild (room temperature).
In addition, synthesis path selectivity with higher, yield, easily conversion are used for industrialization production.
The beneficial effects of the present invention are:
The present invention replaces one of sulfonic group-SO of ICG with modified PEG group3H, so that reaching significant extends ICG
The effect of half-life period in human plasma.To mouse tail vein injection PEG-ICGs and ICG, mouse femoral vein is carried out close red
Outer IIth area fluorescence imaging is simultaneously taken pictures, and according to the ratio of fluorescence intensity, calculates PEG-ICGs and the ICG respective metabolism in blood plasma
Time finds that half-life period extends to 1.12731h~6.2413h by the 0.06421h of unmodified ICG, therefore compared with unmodified
ICG, the ICG being modified with the modified PEG group of molecular weight 500,2000,5000,10000,20000, when being detained in blood
Between be significantly longer, so that it is navigated in angiography, art, various applications such as the detection of tumour and treatment are optimised.
Meanwhile by carrying out absorption spectrum intensity detection to the PEG-ICGs after modification, it is possible to find modify and unmodified
ICG has almost the same spectral absorptive capacity, and in I area (wavelength 650nm-900nm) of near-infrared and II area's (wavelength
Good photoluminescent property 900nm-1200nm) is all had, and consistent with the absorption of unmodified ICG and emission spectrum property.
Thus illustrate, PEG-ICGs has Ith area of near-infrared as ICG and the application value of IIth area imaging.In addition, after tested, institute
State modified PEG-ICGs shown in the PBS (phosphate buffered saline solution) and FBS (fetal calf serum) of pH7.4 it is good steady
It is qualitative, therefore the modified PEG-ICGs of the present invention has the prospect commercially produced and applied.
Detailed description of the invention
Figure 1A is the molecular structural formula of indocyanine green ICG, and Figure 1B is the molecular structure of the modified PEG-ICGs of the present invention
Formula.
Fig. 2A is that the intermediate product ICG-NHS of modification ICG is purified through efficient liquid phase chromatographic analysis and examined under the channel 780nm
Survey absorption peak.
Fig. 2 B, Fig. 2 C, Fig. 2 D, Fig. 2 E, Fig. 2 F are respectively through molecular weight 500,2000,5000,10000,20000
mPEG-NH2Modify ICG resulting PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and
PEG20000-ICG is purified through efficient liquid phase chromatographic analysis and is detected absorption peak under the channel 780nm.
Fig. 3 be 20 μ g/ml of concentration unmodified ICG and modified PEG500-ICG, PEG2000-ICG,
The relative absorbance spectrum of PEG5000-ICG, PEG10000-ICG and PEG20000-ICG in wavelength 600nm~950nm.
Fig. 4 be unmodified ICG and modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG,
Relative intensity of fluorescence of the PEG10000-ICG and PEG20000-ICG in I area (wavelength 650nm-900nm) of near-infrared.
Fig. 5 be unmodified ICG and modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG,
Relative intensity of fluorescence of the PEG10000-ICG and PEG20000-ICG in II area (wavelength 900nm-1200nm) of near-infrared.
Fig. 6 be modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and
PBS (ingredient Na of the PEG20000-ICG in pH7.42HPO4And NaH2PO4) in fluorescence intensity rate of descent change over time song
Line.
Fig. 7 be modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and
PEG20000-ICG fluorescence intensity rate of descent in FBS (fetal calf serum) changes over time curve.
Fig. 8 be unmodified ICG and modified PEG500-ICG, PEG2000-ICG, PEG5000-ICG,
PEG10000-ICG and PEG20000-ICG is pressing 2mgmL-1After Tail Vein injection Mouse, in II area's fluorescence imaging of near-infrared
Photo group.
Fig. 9 is to distinguish the unmodified ICG and modified PEG5000- of tail vein injection to the mouse with tumour
ICG tests the EPR effect of modified PEG5000-ICG in the photo of II area's fluorescence imaging of near-infrared after different time.
Specific embodiment
In order to preferably explain the present invention, in order to understand, with reference to the accompanying drawing, by specific embodiment, to this hair
It is bright to be described in detail.
The present invention mainly modifies blood vessel fluorescence imaging material ICG with modified PEG group, to extend it in blood
Half-life period in liquid.Modified PEG is called modified polyethylene glycol, is repaired by chemical modification group or bio-active group
The PEG of decorations, i.e. routine PEG change its end group, with-OH ,-COOH ,-NH2, methoxyl group, itrile group with bioaffinity
Deng substitution or connection.Modified PEG group refer to modified PEG lose end H atom after be formed by active group.Modification
Property PEG includes but is not limited to mPEG (mono methoxy ether PEG), mPEG-NH2 (mPEG- amido), mPEG-SS (mPEG- succinyl
Imines succinate), mPEG-SC (mPEG-SC), mPEG2-NHS (mPEG2-N- HOSu NHS
Ester), mPEG-SPA (mPEG- succinimide propionic acid acid esters), mPEG-ALD (mPEG- propionic aldehyde), (Malaysia mPEG- mPEG-MAL
Acid imide), HO-PEG-COOH (a- hydroxyl-w- carboxy polyethylene glycol), the mPEG-b-PS (block of polyethylene glycol and polystyrene
Copolymer), mPEG-b-PI (block copolymer of polyethylene glycol and polyisoprene), mPEG-b-PAN (polyethylene glycol and poly- third
The block copolymer of alkene nitrile), mPEG-b-PCL (block copolymer of polyethylene glycol and poly-epsilon-caprolactone), mPEG-b-PMMA it is (poly-
The block copolymer of ethylene glycol and polymethyl methacrylate), α-hydroxyl-PEG- ω-amide, double hydroxyl PEG, HO-
PEG-NH2 (Alpha-hydroxy-omega-amino polyethylene glycol), mPEG-carboxyl (mPEG- carboxyl), mPEG-cyan (mPEG- itrile group)
Deng.Wherein, the molecular weight value range of modified PEG group is 500~20000, preferably 500,2000,5000,10000,
20000。
As the present invention, more preferably embodiment, the molecular structural formula of the indocyanine green through PEG modification are expressed as follows:
Wherein, n=9~450.
Prepare it is above-mentioned through PEG modification indocyanine green method: with warp-CO-NHS ester group modification ICG derivative and
mPEG-NH2For reactant, mPEG-NH2Ester substitution reaction occurs with-CO-NHS in the ICG derivative, sloughs a molecule N- hydroxyl
The novel I CG structural formula C modified by mPEG-NH- is made in succinimide；Its reaction process indicates are as follows:
Preferably, the condition of above-mentioned reaction are as follows: by-NHS modification ICG derivative (structural formula B) respectively with different molecular
The mPEG-NH of amount2The ratio of (structural formula A) 1:5 in molar ratio, in the PBS of pH=7.0~8.0, room temperature shaker reaction 5~
12h is purified through HPLC, obtains PEG-ICG, i.e. product represented by structural formula C.
The present invention ICG (structural formula C) modified through modified PEG group, the residence time is significantly longer in blood, makes
It navigates in angiography, art, and detection and the various applications such as treatment of tumour are optimised.
According to the method described above, specific preparation example and test case are enumerated below.
Preparation example 1
Preparation method: by the ICG derivative of warp-CO-NHS ester group modification, (structural formula B, is purchased fromIntrace Medical SA) with the mPEG-NH of molecular weight 5002The ratio of (structural formula A) 1:5 in molar ratio, in the PBS of pH=7.4, room temperature shaker
12h is reacted, is purified through HPLC, obtains PEG-ICG (structural formula C), as shown in Figure 1B.
Preparation example 2
This example is identical as preparation example 1, the mPEG-NH for the use of molecular weight being about only 20002Participate in reaction.Product is denoted as
PEG2000-ICG。
Preparation example 3
This example is identical as preparation example 1, the mPEG-NH for the use of molecular weight being about only 50002Participate in reaction.Product is denoted as
PEG5000-ICG。
Preparation example 4
This example is identical as preparation example 1, the mPEG-NH for the use of molecular weight being about only 100002Participate in reaction.Product is denoted as
PEG10000-ICG。
Preparation example 5
This example is identical as preparation example 1, the mPEG-NH for the use of molecular weight being about only 200002Participate in reaction.Product is denoted as
PEG20000-ICG。
Test case
1, Product formation characterization test
Below to the resulting PEG500-ICG, PEG2000-ICG of 1~preparation example of preparation example 5, PEG5000-ICG,
PEG10000-ICG and PEG20000-ICG is purified through high performance liquid chromatographs (HPLC), selection splitter (C4Column,
300A, 3.5 μm, 4.6mm × 250mm), absorption peak is detected under the channel 780nm.Program such as table 1:
Table 1:
Referring to fig. 2 shown in A~Fig. 2 F.Reactant ICG-NHS (warp-CO-NHS ester group the modification that wherein Fig. 2A is structural formula B
ICG) HPLC figure, retention time in splitter is 22.4943min, and ICG-NHS has polarity small, and lipophilicity is water-soluble
The features such as property is poor.
The HPLC figure that Fig. 2 B is the PEG500-ICG of preparation example 1, appearance time are 20.7236min earlier than ICG-NHS, are said
Bright PEGylated due to ICG, polarity is opposite to become larger, and water solubility improves, it was demonstrated that mPEG-NH2With ICG successful connection.Due to not with
The mPEG-NH of ICG connection2There is no specific absorption peak, therefore HPLC can't detect mPEG-NH2.And the appearance time of Fig. 2 B earlier than
ICG-NHS, it was demonstrated that mPEG-NH2With ICG successful connection.
The HPLC figure that Fig. 2 C is the PEG2000-ICG of preparation example 2, appearance time be 19.6685min earlier than ICG-NHS and
PEG500-ICG, same explanation is due to mPEG-NH2Molecular weight it is bigger, further polarize ICG, and water solubility improves, so
The appearance time ratio PEG500-ICG of PEG2000-ICG is earlier, it was demonstrated that mPEG-NH2With ICG successful connection.
Fig. 2 D be preparation example 3 PEG5000-ICG figure HPLC figure, appearance time be 19.2725min earlier than ICG-NHS,
PEG500-ICG, PEG2000-ICG similarly prove mPEG-NH2With ICG successful connection.
The HPLC figure that Fig. 2 E is the PEG10000-ICG of preparation example 4, appearance time are 19.6829min earlier than ICG-NHS,
But appearance time is later than PEG5000-ICG, probably due to the mPEG-NH of connection2Molecular weight is excessive, by the shadow of the position in column
It rings, so while water solubility improves but appearance time is but later than PEG5K-ICG.But likewise, not with ICG successful connection
mPEG-NH2It is no specific absorption peak, so the provable mPEG-NH of Fig. 2 E2With ICG successful connection.
The HPLC figure that Fig. 2 F is the PEG20000-ICG of preparation example 5, appearance time 19.4424min, appearance is earlier than ICG-
NHS, appearance time are later than PEG5K-ICG.It is also likely to be since the PEG molecular weight of connection is excessive, so while water solubility improves
But appearance time is but later than PEG5K-ICG.But same reason, the provable mPEG-NH of Fig. 2 F2With ICG successful connection.
It can be just detected by PEG-ICGs only successful connection, and the appearance time of appearance time ratio ICG-NHS is early, says
Bright successful connection, and the PEG-ICGs after successful connection has preferably water solubility compared to ICG-HNS.
2, product spectrum property is tested
To preparation example 1-5 preparation PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and
PEG20000-ICG tests its spectral property (including absorption spectra property and emission spectrum property) respectively, and with it is unmodified
ICG is compared.It specifically operates as follows:
Using UV-Vis-NIR spectrometer cary 5000, surveyed respectively in spectral region 500nm-1000nm ICG,
The spectral absorption intensity of PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG.
It is shown in Figure 3.It wherein aggravates full curve and represents PEG20000-ICG, continuous gray scale line represents ICG, dark exacerbation dotted line generation
Table PEG500-ICG, fine dotted line represent PEG2000-ICG, single dotted broken line represents PEG5000-ICG, and double dot dash line represents
PEG10000-ICG.From the figure 3, it may be seen that modified PEG-ICGs has the spectral absorptive capacity almost the same with unmodified ICG.
Using spectrophotometer Spectronic 200, set exciting light 808nm, test respectively ICG, PEG500-ICG,
PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG are in Ith area of near-infrared (650nm-900nm range
It is interior) relative intensity of fluorescence.Test result is shown in Figure 4.It wherein aggravates full curve and represents PEG20000-ICG, gray scale connects
Continuous line represents ICG, dark aggravates that dotted line represents PEG500-ICG, fine dotted line represents PEG2000-ICG, single dotted broken line represents
PEG5000-ICG, double dot dash line represent PEG10000-ICG.As shown in Figure 4, in Ith area of near-infrared, modified outcome PEG500-
ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG have suitable with unmodified ICG
Good photoluminescent property.As it can be seen that modification of the modified PEG to ICG, do not have an impact photoluminescent property possessed by former ICG.
Using spectrometer NIRQuest512 and CVH100/M, exciting light 808nm is set, tests ICG, PEG500- respectively
ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG are in II area (900nm- of near-infrared
Within the scope of 1200nm) relative intensity of fluorescence.Test result is shown in Figure 5.It wherein aggravates full curve and represents PEG20000-
ICG, continuous gray scale line represent ICG, dark aggravate that dotted line represents PEG500-ICG, fine dotted line represents PEG2000-ICG, single-point is drawn
Line represents PEG5000-ICG, and double dot dash line represents PEG10000-ICG.As shown in Figure 5, in IIth area of near-infrared, modified outcome
PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG have with it is unmodified
The comparable good photoluminescent property of ICG.
Show the ICG after mPEG-NH2 is modified by the test of the above spectral property, has in Ith area of near-infrared and IIth area
Good photoluminescent property, and it is consistent with the absorption of ICG and emission spectrum property.Therefore, PEG-ICGs has comparable close with ICG
The application value in infrared Ith area and the imaging of IIth area.
3, product stability is tested
(1) neutral solution stability test
Take respectively modified outcome PEG500-ICG, PEG2000-ICG, PEG5000-ICG prepared by preparation example 1-5,
PEG10000-ICG and PEG20000-ICG, which is dissolved in the PBS solution of pH7.4, obtains 5 test samples, concentration be 20 micrograms/
mL.PBS buffer solution ingredient is Na2HPO4And NaH2PO4.It is protected from light 5 test samples of standing or more in insulating box, exists respectively
3h, 6h, 9h, 12h, 15h, 18h, 21h, for 24 hours when, test the fluorescence intensity of 5 solution examples, and calculate rate of descent.Test makes
With spectrophotometer Spectronic 200, exciting light 808nm is set.Test results are shown in figure 6.The result shows that removing
The fluorescence intensity decline of PEG500-ICG shows slightly very fast outer in 1~12 hour, other PEG2000-ICG, PEG5000-ICG,
The passage of the fluorescence intensity of PEG10000-ICG and PEG20000-ICG at any time is all in stable state.Thus illustrate, modify
Product PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG are close to neutral ring
In border, stability is preferable.
(2) iii vivo serum stability test
Take respectively modified outcome PEG500-ICG, PEG2000-ICG, PEG5000-ICG prepared by preparation example 1-5,
PEG10000-ICG and PEG20000-ICG, which is dissolved in FBS (fetal calf serum, the SR01C-500 purchased from SAILY BIO), obtains 5
Test sample (concentration is 20 micrograms/mL).
Above 5 test samples are stood in insulating box, respectively 3h, 6h, 9h, 12h, 15h, 18h, 21h, for 24 hours when, survey
The fluorescence intensity of 5 test samples is tried, and calculates rate of descent.Test uses spectrophotometer Spectronic 200, and setting swashs
Shine 808nm.Test results are shown in figure 7.The result shows that the passage of the fluorescence intensity of 5 test samples at any time all in
Highly stable state.Thus illustrate, modified outcome PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG
With PEG20000-ICG in serum, stability is preferable.It is preferable real that result above shows that modified outcome PEG-ICGs has
Border application prospect has commercialization condition.
4, plasma half-life tests
By 18 healthy mouse (20g or so), it is divided into 6 groups, every group 3.6 groups of mouse press the amount of ICG in component respectively
1mg·kg-1(injection volume 0.1-1mgkg-1, concentration containing ICG is 20 micrograms/mL in injection) and tail vein injection
ICG, PEG500-ICG, PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG.Again respectively at
After 3min, 8min, 30min, 120min, 240min, 270min, 600min, by II area's fluorescence imaging of near-infrared, pass through blood vessel
Fluorescence imaging compares the blood clearance rate of 5 kinds of mPEG-ICG of ICG or more.Concrete operation method and calculating are performed as follows:
Imaging photographical condition: being excited by 808nm laser, and 900nm long wave pass filter FELH0900 is by exciting light and sends out
It penetrates light to separate, be taken pictures by the imaging of SWIR near infrared camera.
Mouse is anaesthetized by Animal Anesthesia system VIP3000, five kinds of PEG-ICGs of tail vein injection (PEG500-ICG,
PEG2000-ICG, PEG5000-ICG, PEG10000-ICG and PEG20000-ICG) and ICG after, respectively at the same time
Point 3min, 4min, 5min, 6min, 7min, 8min, 12min carry out II area's fluorescence imaging of near-infrared to mouse femoral vein and clap
According to, and referring to photo group shown in appended Fig. 8.
Calculate ICG or PEG-ICGs half-life period in blood plasma, be by the fluorescence intensity of the vascular site in image photograph with
Leg skin part fluorescence intensity, which is compared, finds out ratio, to reflect the plasma metabolism time of PEG-ICGs and ICG.Its
In, 3min back leg medial vein blood vessel is high-visible after injection for ICG group mouse, and the fluorescence signal of 8min or so vasculature part disappears
It loses, the T of ICG is calculated(1/2)=0.06421h.
PEG500-ICG group mouse after injection 3min back leg medial vein blood vessel be also it is high-visible, 12min or so is glimmering
Optical signal disappears, and the T of PEG500-ICG is calculated(1/2)=0.16121h.It compares for ICG group mouse, PEG500-ICG
The metabolism time of PEG500-ICG, there is no extended in the extreme in group Mice Body.
Fluorescence signal disappears PEG2000-ICG group mouse at 135min or so blood vessel after injection, and T is calculated(1/2)=
1.12731h.Blood residence time than injecting ICG and PEG500-ICG merely extends.
Fluorescence signal disappears PEG5000-ICG group mouse at 240min or so blood vessel after injection, and T is calculated(1/2)=
2.417h.Blood residence time than injecting ICG, PEG500-ICG and PEG2000-ICG merely extends.
Fluorescence signal is still faint as it can be seen that calculating at 270min or so blood vessel after injection for PEG10000-ICG group mouse
To T(1/2)=2.6667h.When blood than injecting ICG, PEG500-ICG, PEG2000-ICG and PEG5000-ICG merely is detained
Between extend.
5h or so fluorescence signal can also detect PEG20000-ICG group mouse after injection, can continue until 10h.It calculates
To T(1/2)=6.2413h.Than injecting ICG, PEG500-ICG, PEG2000-ICG, PEG5000-ICG and PEG10000- merely
The blood residence time of ICG extends.
Thus illustrate, modify PEG2000-ICG, PEG5000-ICG, PEG10000- that ICG is obtained by the method for the invention
ICG and PEG20000-ICG, relatively unmodified ICG, residence time is extended significantly in animal body blood plasma.To logical
The modification to ICG is crossed, it is made to navigate in angiography, art, detection and the various applications such as treatment of tumour are optimised.
5, EPR effect test in animal body
EPR effect, the i.e. high-permeability of solid tumor and retention effect (enhanced permeability and
retention effect).It specifically refers to relative to normal tissue, the molecule or particle of certain sizes tend to
It is gathered in the property of tumor tissues.Main cause is that microvascular endothelial gap in normal tissue is fine and close, structural integrity, macromolecular
It is not easy with lipid granule through vascular wall, and solid tumor mass medium vessels are abundant, vascular wall gap is wider, poor structural integrity,
Lymphatic return missing causes macromolecular substance and lipid granule to have selective high-permeability and anelasticity, this phenomenon quilt
The referred to as high-permeability and retention effect of solid tumor mass, abbreviation EPR effect.It measures drug or whether diagnostic materials is suitable for use in
The detection of tumour, an important index are exactly EPR effect.Here, the EPR effect to PEG-ICGs prepared by the present invention carries out
Test.Test method is as follows:
Prepare skov-3 tumour cell (being purchased from EK-Bioscience) and weight is mouse 2 of 300g/ only, by every
2000000 cells of tumour, subcutaneous injection, after 3-4 weeks, tumor size to 5mm-10mm.By 2 mouse with tumour by moving
Object anesthesiaing system VIP3000 anesthesia, with containing ICG meter (since ICG with PEG5000-ICG molecular weight is different, 1mg herein
kg-1It is calculated with the amount of contained ICG；When injection, ICG and PEG5000-ICG are dissolved in the PBS of pH7.4), every 1mg
kg-1Tail vein injection ICG and PEG5000-ICG.Inject ICG mouse 3min, 4h and 6h by II area's fluorescence of near-infrared at
Picture, the mouse for injecting PEG5000-ICG pass through II area's fluorescence imaging of near-infrared in 3min, 12h and 48h.Photographical condition is imaged: by
The excitation of 808nm laser, 900nm long wave pass filter FELH0900 separates exciting light and transmitting light, by SWIR near-infrared phase
Machine imaging is taken pictures.It takes a picture as shown in Figure 9.
Wherein, inject the mouse of PEG5000-ICG, blood vessel is very clear after 3min, at tumour, 12h when tumour at fluorescence
Signal is obvious, and there are very strong EPR effect, imaging when than the mouse 3min of injection IEG is relatively sharp.Inject PEG5000-
The imaging of the mouse 3min of the close injection IEG of imaging after the mouse 48h of ICG.It can be seen that PEG-ICGs of the invention has
Very strong EPR effect, can be applied to the detection to animal body tumour.
Claims (7)
1. a kind of modified indocyanine green is to be repaired indocyanine green molecular structure two sulfonic one of sulfonic groups
Replaced decorations property PEG group.
2. the indocyanine green according to claim 1 modified through PEG, which is characterized in that the modified PEG group is choosing
From mPEG, mPEG-NH2, mPEG-SS, mPEG-SC, mPEG2-NHS, mPEG-SPA, mPEG-ALD, mPEG-MAL, HO-PEG-
COOH、mPEG-b-PS、mPEG-b-PI、mPEG-b-PAN、mPEG-b-PCL、mPEG-b-PMMA、α-hydroxyl-PEG-ω-
The end group of any modified PEG in amide, HO-PEG-OH, HO-PEG-NH2, mPEG-carboxyl and mPEG-cyan
Missing H is formed by group.
3. the indocyanine green according to claim 1 modified through PEG, which is characterized in that point of the modified PEG group
Son amount value range is 500~20000.
4. the indocyanine green according to claim 3 modified through PEG, which is characterized in that point of the modified PEG group
Son amount is 500,2000,5000,10000 or 20000.
5. the indocyanine green according to claim 1 modified through PEG, which is characterized in that the modified indocyanine green
Molecular structural formula be expressed as follows:
Wherein, n=9~450.
6. the molecular structural formula of a kind of method for preparing modified indocyanine green, the modified indocyanine green indicates such as
Under:
Wherein n=9~450；The described method includes:
The ICG derivative and mPEG-NH modified with warp-CO-NHS ester group2For reactant, mPEG-NH2With in the ICG derivative-
Ester substitution reaction occurs for CO-NHS, sloughs a molecule n-hydroxysuccinimide, and the novel I CG modified by mPEG-NH- is made and ties
Structure formula C；Its reaction process indicates are as follows:
7. according to the method described in claim 6, it is characterized in that, the reaction condition of the method are as follows: the warp-CO-NHS ester
The ICG derivative and mPEG-NH of base modification2The ratio mixing of 1:5 in molar ratio, in the PBS of pH=7.0~8.0, room temperature is shaken
Bed 5~12h of reaction, is made a kind of novel I CG modified through mPEG-NH-.
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Application publication date: 20181221 |