CN116903782A - Polyacrylate fluorescent probe based on benzothiadiazole, synthesis method and application thereof in monitoring lipid droplets - Google Patents
Polyacrylate fluorescent probe based on benzothiadiazole, synthesis method and application thereof in monitoring lipid droplets Download PDFInfo
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- CN116903782A CN116903782A CN202310832930.2A CN202310832930A CN116903782A CN 116903782 A CN116903782 A CN 116903782A CN 202310832930 A CN202310832930 A CN 202310832930A CN 116903782 A CN116903782 A CN 116903782A
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- 150000002632 lipids Chemical class 0.000 title claims abstract description 35
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 24
- 229920000058 polyacrylate Polymers 0.000 title claims abstract description 7
- FNQJDLTXOVEEFB-UHFFFAOYSA-N 1,2,3-benzothiadiazole Chemical compound C1=CC=C2SN=NC2=C1 FNQJDLTXOVEEFB-UHFFFAOYSA-N 0.000 title claims abstract description 6
- 239000005964 Acibenzolar-S-methyl Substances 0.000 title claims abstract description 6
- 238000001308 synthesis method Methods 0.000 title claims description 7
- 238000012544 monitoring process Methods 0.000 title claims description 6
- 239000000523 sample Substances 0.000 claims abstract description 80
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 238000003384 imaging method Methods 0.000 claims abstract description 7
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 53
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 26
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- 229940126214 compound 3 Drugs 0.000 claims description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 229940125782 compound 2 Drugs 0.000 claims description 13
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 10
- 238000002189 fluorescence spectrum Methods 0.000 claims description 10
- VHRYZQNGTZXDNX-UHFFFAOYSA-N methacryloyl chloride Chemical compound CC(=C)C(Cl)=O VHRYZQNGTZXDNX-UHFFFAOYSA-N 0.000 claims description 9
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- PNYGERBROPJVCX-UHFFFAOYSA-N 2-tridecanethioylsulfanylpropanoic acid Chemical compound CCCCCCCCCCCCC(=S)SC(C)C(O)=O PNYGERBROPJVCX-UHFFFAOYSA-N 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- -1 2- [ (7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino ] ethyl Chemical group 0.000 claims description 4
- OYABVKXAVFCRHU-UHFFFAOYSA-N 4-bromo-7-nitro-2,1,3-benzothiadiazole Chemical class [O-][N+](=O)C1=CC=C(Br)C2=NSN=C12 OYABVKXAVFCRHU-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- YYIBRXXNWYMKRO-UHFFFAOYSA-N 2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethanol Chemical compound OCCNC1=CC=C([N+]([O-])=O)C2=NON=C12 YYIBRXXNWYMKRO-UHFFFAOYSA-N 0.000 claims description 2
- 150000002169 ethanolamines Chemical class 0.000 claims description 2
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- 230000002194 synthesizing effect Effects 0.000 claims 1
- 230000008859 change Effects 0.000 abstract description 4
- 238000001917 fluorescence detection Methods 0.000 abstract description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 40
- 210000004027 cell Anatomy 0.000 description 21
- 229920000642 polymer Polymers 0.000 description 16
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 15
- 239000002904 solvent Substances 0.000 description 14
- 239000011550 stock solution Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 11
- 230000008045 co-localization Effects 0.000 description 8
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- 239000012224 working solution Substances 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 230000005284 excitation Effects 0.000 description 7
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 239000002798 polar solvent Substances 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
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- 239000000203 mixture Substances 0.000 description 3
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- 230000008685 targeting Effects 0.000 description 3
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
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- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
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- 230000001413 cellular effect Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
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- OBISXEJSEGNNKL-UHFFFAOYSA-N dinitrogen-n-sulfide Chemical compound [N-]=[N+]=S OBISXEJSEGNNKL-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 238000002347 injection Methods 0.000 description 1
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- 230000002452 interceptive effect Effects 0.000 description 1
- 210000005061 intracellular organelle Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005892 protein maturation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000028503 regulation of lipid metabolic process Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
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- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- WHRNULOCNSKMGB-UHFFFAOYSA-N tetrahydrofuran thf Chemical compound C1CCOC1.C1CCOC1 WHRNULOCNSKMGB-UHFFFAOYSA-N 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- VLLMWSRANPNYQX-UHFFFAOYSA-N thiadiazole Chemical compound C1=CSN=N1.C1=CSN=N1 VLLMWSRANPNYQX-UHFFFAOYSA-N 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
- C08F220/285—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety
- C08F220/286—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety and containing polyethylene oxide in the alcohol moiety, e.g. methoxy polyethylene glycol (meth)acrylate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a polyacrylate fluorescent probe based on benzothiadiazole, which is NBMA-co-PEGMA400, and the NBMA-co-PEGMA400 probe can be applied to dynamic sensing detection of lipid droplets in a biological system; the sensing detection comprises fluorescence detection, cell imaging and the like; the fluorescent probe is sensitive to polar environment and has good lipophilicity to THF; the fluorescence probe has small change of fluorescence intensity in the environment with pH=0.998-12.072, and has good pH resistance.
Description
Technical Field
The invention relates to a polymer fluorescent probe of a polyacrylate marked intracellular lipid drop based on diazosulfide, and a spectrum test and a cell imaging; belongs to the technical field of biological medicine.
Background
Lipid droplets are a dynamic intracellular organelle, also known as liposomes, oil bodies or fat bodies, which are lipid-rich organelles that regulate the storage and hydrolysis of neutral lipids, mainly in adipose tissue. They can also be used as reservoirs of cholesterol and acylglycerols for film formation and maintenance. Lipid droplets consist of a neutral lipid core, consisting essentially of Triacylglycerols (TAGs) surrounded by a phospholipid monolayer and cholesterol esters. The surface of lipid droplets is modified by a number of proteins involved in the regulation of lipid metabolism. Lipid droplets play a central role in energy homeostasis and lipid metabolism. In addition, lipid droplets aid in cellular protein transport and protein maturation. Lipid droplets are involved in a variety of physiological processes within cells, and their dysfunction is associated with a variety of diseases, so lipid droplet studies are of great importance for the etiology of a variety of diseases and the prevention. The advent and development of fluorescent probe technology has attracted considerable attention to many researchers so far. Compared with the traditional detection method, the fluorescence sensing technology has good timeliness and convenience. Accordingly, a number of fluorescent probes for specific labeled lipid droplets have been developed. In addition, specific probes with specific properties have been developed to study lipid droplet function and disease correlation. Because the lipid droplet environment provides a more hydrophobic and more viscous differential environment, if a fluorescent probe that can distinguish between the internal and external environments of a lipid droplet could be found, a new means would be provided for further investigation of the pathogenic mechanism and therapeutic regimen of the lipid droplet.
Although some lipid droplet fluorescent probes are currently commercially available, polymer-based lipid droplet probes have few reports, and most of the known small molecule probes have poor photostability, including emission of innate biomolecules (autofluorescence).
Therefore, the invention designs a polymer probe for dynamically monitoring intracellular lipid droplets, which has the characteristics of strong specificity, good water solubility, lower biotoxicity and stable pH.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a polyacrylate fluorescent probe based on benzothiadiazole, a synthesis method and application thereof in monitoring lipid drops, and the following aims are achieved: sensitive to polar environment, wide pH resistant range, strong anti-ion interference capability, good light stability under environments with different polarities, good water solubility, low biotoxicity and strong specificity.
In order to solve the technical problems, the invention adopts the following technical scheme:
a benzothiadiazole-based polyacrylate fluorescent probe, the structural formula of which is as follows:
n=7。
the synthesis method of the fluorescent probe comprises the steps of dissolving a compound 3, PEGMA400, azodiisobutyronitrile, (2- [ (dodecyl thio-carbonyl) thio ] propionic acid) in tetrahydrofuran, deoxidizing a reaction system, reacting for 23-25h at 69-71 ℃ and purifying to obtain the fluorescent probe NBMA-co-PEGMA400.
The mol ratio of the compound 3 to the PEGMA400 is 0.05:0.9-1.1; the mol ratio of the compound 3 to the azodiisobutyronitrile is 9.5-10.5:1; the molar ratio of the compound 3 to the (2- [ (dodecyl thio-carbonyl) thio ] propionic acid is 1.8-2.2:1; the mass volume ratio of the compound 3 to the tetrahydrofuran is 5-5.2 mg/1 mL.
The preparation method of the compound 3 comprises the following steps: compound 2 was dissolved in dichloromethane and triethylamine was added to it at N 2 Under the conditions of environment and ice water bath, slowly adding the methacryloyl chloride into a reaction system, stirring for 0.9-1.1h, then transferring into normal temperature for reaction 23-25h, and extracting, drying and purifying to obtain the compound 3.
The mol ratio of the compound 2 to the methacryloyl chloride is 1:2.9-3.1; the volume ratio of the dichloromethane to the methacryloyl chloride is 39-41:1; the volume ratio of the triethylamine to the methacryloyl chloride is 1:0.9-1.1.
The preparation method of the compound 2 comprises the following steps: 4-bromo-7-nitro-benzo [1,2,5]Thiadiazoles and ethanolamines are dissolved in ethanol, at N 2 And (3) carrying out reflux reaction for 4.3-4.7h at 89-91 ℃ in the environment, and purifying to obtain the compound 2.
The molar ratio of the 4-bromo-7-nitro-benzo [1,2,5] thiadiazole to the ethanolamine is 1:1.1-1.3; the mass volume ratio of the ethanolamine to the ethanol is 1mg:1.6-1.7mL.
The compound 2 is (2- ((7-nitrobenzo [ c ] [1,2,5] oxadiazol-4-yl) amino) ethanol).
The compound 3 is (2- [ (7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino ] ethyl 2-methyl-2-acrylate).
The fluorescent probe is applied to monitoring lipid droplets, and the probe carries out specific dynamic sensing detection on the lipid droplets, wherein the sensing detection refers to fluorescence spectrum detection or fluorescent cell imaging.
The synthesis route of the probe NBMA-co-PEGMA400 is as follows:
compared with the prior art, the invention has the following beneficial effects:
(1) The invention discloses application of an NBMA-co-PEGMA400 probe: the fluorescent probe can be applied to dynamic sensing detection of lipid droplets in a biological system; the sensing detection comprises fluorescence detection, cell imaging and the like.
(2) The fluorescent probe is sensitive to polar environment and has good lipophilicity to THF.
(3) The fluorescence probe has small change of fluorescence intensity under the environment of pH=0.998-12.072, and has good pH resistance.
(4) The fluorescent probe has good light stability, and has good light stability in toluene or PBS.
(5) The fluorescent probe has good water solubility and low biotoxicity, and the cell survival rate is more than 90% when the probe and cells are incubated together.
(6) The fluorescent probe provided by the invention has the characteristics of reliability and accuracy in detecting intracellular lipid droplets, has good targeting property on LDs, and has a lipid droplet marking dye Nile Red and a Pearson co-localization coefficient of the probe on Hela cells of 0.89.
Drawings
FIG. 1 is a diagram of Compound 2 in example 1 1 H NMR spectrum;
FIG. 2 is a diagram of Compound 3 of example 1 1 H NMR spectrum;
FIG. 3 is a schematic illustration of the NBMA-co-PEGMA400 compound of example 1 with each monomer 1 H NMR contrast profile;
FIG. 4 is an ultraviolet absorbance spectrum of probe NBMA-co-PEGMA400 in solvents of different polarities;
FIG. 5 is a normalized spectrum of fluorescence emission of probe NBMA-co-PEGMA400 in common solvents of different polarities;
FIG. 6 is a fluorescence emission spectrum of a mixed solution of probe NBMA-co-PEGMA400 in different proportions of THF and PBS;
FIG. 7 is a fluorescence spectrum of probe NBMA-co-PEGMA400 in a mixed solution of THF and PBS at different pH;
FIG. 8 is probe NBMA-co-PEGMA400 selectivity histogram data;
wherein 1: blanc; 2: HS (HS) - ；3：NO 2 - ；4：CO 3 2- ；5：TBHP；6：H 2 O 2 ；7：CI - ；8：SO 4 2- ；9：Ca 2+ ；10：HSO 3 - ；11：CYS；12：HCO 3 - ；13：Mg 2+ ；14：Na + ；
FIG. 9 is a light stability test chart of probe NBMA-co-PEGMA 400;
FIG. 10 is a graph of water solubility test and fit of probe NBMA-co-PEGMA 400;
FIG. 10a is a water solubility test chart; FIG. 10b is a graph of fit;
FIG. 11 is a SEC elution profile of probe NBMA-co-PEGMA 400;
FIG. 12 is a histogram of MTT cytotoxicity test of probe NBMA-co-PEGMA 400;
FIG. 13 is a lipid drop co-localization test pattern for probe NBMA-co-PEGMA400 and Nile Red applications;
wherein fig. 13 a) bright field of HeLa cells; b) NBMA-co-PEGMA400 (10 [ mu ] g/mL) stains yellow channel; c) Nile Red (5.0 [ mu ] M) dyeing Red channelThe method comprises the steps of carrying out a first treatment on the surface of the d) A combined image of (b) and (c); e) An intensity scatter plot of (b) and (c); f) Fluorescent co-localization curves for b) and c). Yellow channel NBMA-co-PEGMA400: excitation wavelength lambda ex =405 nm, acquisition wavelength 440-480 nm. The Nile Red channel: excitation wavelength lambda ex =560 nm, the collection wavelength is 570-610 nm.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples, in which the numbers of the compounds correspond to the numbers of the compounds in the above-described schemes.
Example 1: synthesis of the Probe Compound NBMA-co-PEGMA400:
compound 1 (4-bromo-7-nitro-benzo [1,2, 5)]Thiadiazole) (0.5 mmol, 130.05 mg) and ethanolamine (0.6 mmol, 36.8 mg) were dissolved in 62. 62 mL ethanol in N 2 The reaction was refluxed at 90℃for 4.5h under ambient conditions. The reaction product was purified by column chromatography with eluent (petroleum ether: ethyl acetate) configured (V: v=10:1) to give pure compound 2 (2- ((7-nitrobenzo [ c)][1,2,5]Oxadiazol-4-yl) amino) ethanol) shows yellow fluorescence under 365 nm uv lamps. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.68 – 8.42 (m, 1H), 6.64 (d,J= 9.1 Hz, 1H), 4.95 (t,J= 5.6 Hz, 1H), 3.69 (q,J= 5.7 Hz, 1H), 3.56 (s, 1H)。
Compound 2 (2- ((7-nitrobenzo [ c)][1,2,5]Oxadiazol-4-yl) amino) ethanol) (0.5 mmol,120 mg) was dissolved in DCM (dichloromethane) (6 mL) and TEA (triethylamine) (150. Mu.L) was added to the solution in N 2 Methacryloyl chloride (1.5 mmol, 150. Mu.L) was slowly added by injection to the reaction system under ambient conditions and ice water bath conditions, stirred for 1h, and then transferred to ambient temperature for reaction 24 h. The reaction product is treated with distilled water andCH 2 Cl 2 (dichloromethane) extraction, drying over anhydrous sodium sulfate, removal of solvent by rotary evaporator, and purification of the reaction product by column chromatography with (V: v=10:1) eluent (petroleum ether: ethyl acetate) gives pure compound 3 (NBMA) (2- [ (7-nitro-2, 1, 3-benzooxadiazol-4-yl) amino group]Ethyl 2-methyl-2-acrylate) shows yellow fluorescence under 365 nm uv lamp. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.79 (t,J= 6.3 Hz, 1H), 8.63 (d,J= 9.0 Hz, 1H), 6.72 (d,J= 9.1 Hz, 1H), 6.01 (s, 1H), 5.66 (t,J= 1.7 Hz, 1H), 4.37 (t,J= 5.4 Hz, 2H), 3.83 (d,J= 6.1 Hz, 2H), 1.83 (s, 3H)。
The compounding monomer compound 3 (2- [ (7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino group]Ethyl 2-methyl-2-acrylate) (0.05 mmol, 15.4 mg) with chemical 4PEGMA 400 Poly (ethylene glycol) methacrylate) having a molecular weight of 400 (1 mmol, 400 mg), AIBN (azobisisobutyronitrile) (0.005 mmol,0.8 mg), CTA (2- [ (dodecylthiocarbonyl) thio]Propionic acid) (0.025 mmol, 10.1. 10.1 mg) was dissolved in THF (tetrahydrofuran) (3 mL), and the mixture was introduced into a 25 mL pressure-resistant tube to continuously introduce N into the reaction system 2 Bubbling for 30min, removing O 2 Reaction at 70℃for 24 h with 1000 g mol -1 And (3) dialyzing 72-h by a molecular weight cut-off membrane, and purifying to obtain the polymer probe NBMA-co-PEGMA400.
Example 2: ultraviolet absorption spectrum of probe NBMA-co-PEGMA400 in common polar solvent
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL. The stock solution was diluted with common organic solvent and PBS (0.1 mol/L, ph=7.4) to 30 μg/mL working solution for uv absorption testing. As shown in fig. 4, the maximum absorption peak of the polymer probe was found to be around 450 and nm, and there was only a slight change in the above-mentioned polar solvent, which means that the dipole moment of the probe in the ground state was less affected by different solvents. The solvents with absorption values from high to low were PBS, DMSO, etOH, DMF, dioxane, tol, etOAc, CH 3 CN，MeOH，THF。
Example 3: fluorescence emission spectrum of probe NBMA-co-PEGMA400 in common polar solvent
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL. Stock solution was diluted with common organic solvents to 30 μg/mL working solution and the fluorescence emission intensity of the probes at different solvents was tested at an excitation wavelength of 460 nm. As shown in FIG. 5, we normalized the fluorescence spectrum of the probe to explore the relationship between the emission peak of the probe and the polarity of the solvent in different solvents, and the legend orders the polarities of six common solvents in order from large to small. The normalized fluorescence spectrum is found to have a certain red shift phenomenon along with the increase of the polarity of the solvent, and meanwhile, the color of the solvent is changed from green (tolene-6) of Toluene to yellow (DMSO-1) of dimethyl sulfoxide, which indicates that the polymer probe has strong polarity dependence; the corresponding solvents for the graph of FIG. 5 are Toluene-6, dioxane-5, etOAc-4, CH, respectively, from left to right 3 CN-3，MeOH-2，DMSO-1。
Example 4: fluorescence emission spectra of probe NBMA-co-PEGMA400 in Tetrahydrofuran (Tetrahydrofuran) and PBS mixed solutions in different proportions
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL. A series of THF-PBS mixed solutions with equal gradient volume ratio are prepared, and the stock solution is diluted into 30 mug/mL working solution by polar solvents with different proportions of 2mL, so that fluorescence emission is detected by excitation wavelength 460 nm. As shown in FIG. 6, the fluorescence intensity of the polymer probe is rapidly increased along with the increase of the THF content in the THF-PBS system in the legend, and the probe has only weak fluorescence emission in the pure PBS system, further the phenomenon that the fluorescence intensity of the probe is enhanced along with the decrease of the PBS content in the mixed system, namely the relative decrease of the polarity in the system, in the binary mixed system is further shown, and the probe is sensitive to polar environment and simultaneously shows good lipophilicity of the probe to THF organic solvents.
The curves in FIG. 6 correspond from bottom to top to the THF-PBS systems of 0% THF, 10% THF, 20% THF, 30% THF, 40% THF, 50% THF, 60% THF, 70% THF, 80% THF, 90% THF, 95% THF, 99% THF, respectively.
Example 5: fluorescence spectrum test of probe NBMA-co-PEGMA400 under different pH environments
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL. A series of mixed solutions of THF/PBS (V/v=1:1) at different pH were prepared, the stock solution and 2mL mixed solution were prepared as 30 μg/mL working solution, and the fluorescence emission profile was detected. As shown in fig. 7, the maximum emission peak of the probe in the ph=0.998 to 12.072 environment was measured at an excitation wavelength of 460nm, and a dot line graph was drawn with the pH value as the abscissa and the emission peak at 520nm as the ordinate. The detection of the fluorescence intensity of the polymer probe in different pH environments shows that the fluorescence intensity of the probe is hardly affected in a wider pH range, and the polymer probe NBMA-co-PEGMA400 shows better pH resistance and provides theoretical support for the subsequent application of the polymer probe in complex biological systems.
Example 6: probe NBMA-co-PEGMA400 Selective histogram data
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL. mu.L of stock solution was placed in 2mL of a mixture of PBS (0.1 mol/L, pH=7.4), and 20. Mu.L of a standard solution of competitor molecule (10. Mu.M) was added, respectively. At 520nm excitation wavelength, the fluorescence emission spectrum change of the solution is immediately detected, and as can be seen from FIG. 8, other interfering ions 2, HS - ；3，NO 2 - ；4，CO 3 2- ；5，TBHP；6，H 2 O 2 ；7，CI - ；8，SO 4 2- ；9，Ca 2+ ；10，HSO 3 - ；11，CYS；12，HCO 3 - ；13，Mg 2+ ；14Na + Little influence on the fluorescence of NBMA-co-PEGMA400 compound, and shows that the polymer probe is less influenced by environmental factors and has stronger anti-interference capability.
Example 7: light stability test of probe NBMA-co-PEGMA400
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL. Stock solutions were prepared with Toluene (tolene) and PBS (0.1 mol/L, ph=7.4) as 30 μg/mL working solutions, respectively. The working solution is continuously tested in 4500 s under 520nm excitation wavelength, as shown in fig. 9, the fluorescence kinetic spectrogram of the probe in a pure solvent (2 mL) system with two different polarities is not greatly fluctuated, which indicates that the probe can keep stable fluorescence emission in two extreme polarity differentiation solvents, and has good light stability. The upper was 30. Mu.g/mL working solution in Toluene (Toluene), and the lower was 30. Mu.g/mL working solution in PBS.
Example 8: probe NBMA-co-PEGMA400 water solubility test and fitting curve thereof
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL. Different volumes of probe stock were mixed with 20 μlpbs (0.1 mol/L, ph=7.4) to prepare test solutions of different concentration gradients with a final volume of 2 mL. As can be seen from FIG. 10, the absorbance of the polymer probe increases with increasing probe concentration, and the absorbance is linearly related to the probe concentration within 300. Mu.g/mL, indicating that the water solubility of the polymer probe NBMA-co-PEGMA400 is excellent. (the probe concentration is 10 mug/mL-300 mug/mL from bottom to top in sequence).
Example 9: probe NBMA-co-PEGMA400 polymerization degree test
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in chromatographically pure Tetrahydrofuran (THF), and the molecular weight and molecular weight distribution were measured by DAWNEOS gel permeation chromatography-multi-angle laser light scattering combined instrument (SEC-MALLS) of Wyatt, U.S. A., MZ 10 3 +MZ 10 4 Chromatographic column, chromatographic grade THF as mobile phase, flow rate 0.5 mL/min,25 ℃. The test results fig. 11 and table 1 show that the polymer probes were unimodal, and were in normal distribution, indicating that the polymer structure was single and uniform. The number average molecular weight mn=2220, the weight average molecular weight mw=5608 and the molecular weight distribution of 1.537.
TABLE 1 molecular weight distribution data for Polymer probes NBMA-co-PEGMA400
Example 10: probe NBMA-co-PEGMA400 MTT cytotoxicity test
The probe NBMA-co-PEGMA400 prepared in example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 1 mg/mL.
Living cell HeLa was cultured in DMEM medium containing fetal bovine serum (10 vol%, FBS), penicillin (100U/mL) and streptomycin (100. Mu.g/mL) at 37℃under an atmosphere containing 5% carbon dioxide and 95% air for 3 days to give HeLa cell culture broth having a cell density of 1X 10 5 /mL；
100. Mu.L of HeLa cell culture was inoculated into 96-well plates, and probe NBMA-co-PEGMA400 was diluted with a mixture of 99.9% DMEM and 0.1% DMSO by volume, and added to the 96-well plates so that final concentrations of the probe were 0, 2,5, 10, 20, 30. Mu.g/mL, respectively, in each well.
Subsequently, the cells were co-cultured at 37℃for 24 hours in an atmosphere of carbon dioxide (5%) and air (95%). HeLa cells were then washed with PBS buffer, then 200. Mu.L of DMEM medium was added to each well, then 50. Mu.L of MTT (5 mg/mL) was added to each well, and incubation was continued for 4 hours in an environment of 37℃carbon dioxide (5%) and air (95%).
The culture medium and MTT in the wells are discarded, 150 μl of DMSO is added to each well, and the wells are shaken on a shaker for 30min at low speed to dissolve the crystallized product sufficiently to dissolve the residual MTT-formazan crystals.
Yellow MTT is reduced to purple formazan in living cells. The absorbance of the solution was measured at 570 nm using a microplate reader. The absorbance values of the blank control group are firstly measured, then the absorbance values of the cell groups with the final concentrations of 0, 2,5, 10, 20 and 30 mug/mL of NBMA-co-PEGMA400 are sequentially measured, and the ratio of the measured absorbance to the absorbance of the blank control group is measured before, so that the cell survival rate is measured.
Assuming 100% viability of cells from the blank group without NBMA-co-PEGMA400,
as can be seen from FIG. 12, the cell viability was above 90% with increasing probe concentration, indicating that the less biotoxic probes are suitable for cell imaging.
Example 11: co-localization test for probe NBMA-co-PEGMA400 and Nile Red applications
We performed co-localization experiments on HeLa cells using the commercially available lipid droplet marker dye Nile Red and the probe NBMA-co-PEGMA400 at different emission wavelength ranges.
The culture method of HeLa cells was the same as in example 10 to obtain a cell culture solution.
Taking 2mL of cell culture solution, respectively adding 20 mu L of probe with final concentration of 10 mu g/mL or 5 mu L of NileRed dye with final concentration of 5.0 mu M, and carrying out CO at 37 ℃ and 5 percent 2 The incubation was carried out for 30min and the lipid drop co-localization test was performed.
Cells were washed 3 times with PBS buffer before imaging to remove residual probes or dyes.
As expected, cells stained with probe NBMA-co-PEGMA400 showed clear spherical spots (FIG. 13 b) and overlapped well with commercial lipid droplet targeting dye (FIG. 13 d), and the Pearson co-localization coefficient was calculated to be 0.89 for both. The co-localization profile and scatter plot included in the analysis results (FIG. 13e, f) also well supported good targeting of the probe NBMA-co-PEGMA400 to Lipid Droplets (LDs).
Claims (10)
1. A polyacrylate fluorescent probe based on benzothiadiazole is characterized in that: the structural formula of the fluorescent probe is as follows:
where n=7.
2. The method for synthesizing a fluorescent probe according to claim 1, wherein: the synthesis method comprises the steps of dissolving a compound 3, PEGMA400, azodiisobutyronitrile, (2- [ (dodecyl thio-carbonyl) thio ] propionic acid) in tetrahydrofuran, deoxidizing a reaction system, reacting for 23-25h at 69-71 ℃ and purifying to obtain a fluorescent probe NBMA-co-PEGMA400.
3. The synthesis method according to claim 2, characterized in that: the mol ratio of the compound 3 to the PEGMA400 is 0.05:0.9-1.1; the mol ratio of the compound 3 to the azodiisobutyronitrile is 9.5-10.5:1; the molar ratio of the compound 3 to the (2- [ (dodecyl thio-carbonyl) thio ] propionic acid is 1.8-2.2:1; the mass volume ratio of the compound 3 to the tetrahydrofuran is 5-5.2 mg/1 mL.
4. The synthesis method according to claim 2, characterized in that: the preparation method of the compound 3 comprises the following steps: compound 2 was dissolved in dichloromethane and triethylamine was added to it at N 2 Under the conditions of environment and ice water bath, slowly adding the methacryloyl chloride into a reaction system, stirring for 0.9-1.1h, then transferring into normal temperature for reaction 23-25h, and extracting, drying and purifying to obtain the compound 3.
5. The method of synthesis according to claim 4, wherein: the mol ratio of the compound 2 to the methacryloyl chloride is 1:2.9-3.1; the volume ratio of the dichloromethane to the methacryloyl chloride is 39-41:1; the volume ratio of the triethylamine to the methacryloyl chloride is 1:0.9-1.1.
6. The method of synthesis according to claim 4, wherein: the preparation method of the compound 2 comprises the following steps: 4-bromo-7-nitro-benzo [1,2,5]Thiadiazoles and ethanolamines are dissolved in ethanol, at N 2 And (3) carrying out reflux reaction for 4.3-4.7h at 89-91 ℃ in the environment, and purifying to obtain the compound 2.
7. The method of synthesis according to claim 6, wherein: the molar ratio of the 4-bromo-7-nitro-benzo [1,2,5] thiadiazole to the ethanolamine is 1:1.1-1.3; the mass volume ratio of the ethanolamine to the ethanol is 1mg:1.6-1.7mL.
8. The synthetic method according to any one of claims 4 to 6, wherein: the compound 2 is (2- ((7-nitrobenzo [ c ] [1,2,5] oxadiazol-4-yl) amino) ethanol).
9. The synthetic method according to any one of claims 2 to 4, wherein: the compound 3 is (2- [ (7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino ] ethyl 2-methyl-2-acrylate).
10. Use of a fluorescent probe according to claim 1 for monitoring lipid droplets, characterized in that: the probe carries out specific dynamic sensing detection on lipid droplets, and the sensing detection refers to fluorescence spectrum detection or fluorescent cell imaging.
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| CN107400102A (en) * | 2017-07-20 | 2017-11-28 | 合肥工业大学 | A kind of fluorescence dichroic dye of novel polymerizable as well as preparation method and application thereof |
| CN114621159A (en) * | 2022-04-06 | 2022-06-14 | 电子科技大学 | Diazosulfide-based fluorescent material, fluorescent polymer, fluorescent nanoparticles, and preparation method and application thereof |
| CN115477725A (en) * | 2022-08-23 | 2022-12-16 | 齐鲁工业大学 | Polymer fluorescent probe for dynamically monitoring lipid droplets, synthesis method and application thereof |
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| US20140342390A1 (en) * | 2011-12-21 | 2014-11-20 | The University Of Tokyo | Temperature-sensitive fluorescent probe for introduction into cell |
| CN107400102A (en) * | 2017-07-20 | 2017-11-28 | 合肥工业大学 | A kind of fluorescence dichroic dye of novel polymerizable as well as preparation method and application thereof |
| CN114621159A (en) * | 2022-04-06 | 2022-06-14 | 电子科技大学 | Diazosulfide-based fluorescent material, fluorescent polymer, fluorescent nanoparticles, and preparation method and application thereof |
| CN115477725A (en) * | 2022-08-23 | 2022-12-16 | 齐鲁工业大学 | Polymer fluorescent probe for dynamically monitoring lipid droplets, synthesis method and application thereof |
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