CN112370533B - Bioluminescent probe capable of imaging FAP for long time and application thereof - Google Patents
Bioluminescent probe capable of imaging FAP for long time and application thereof Download PDFInfo
- Publication number
- CN112370533B CN112370533B CN202010860240.4A CN202010860240A CN112370533B CN 112370533 B CN112370533 B CN 112370533B CN 202010860240 A CN202010860240 A CN 202010860240A CN 112370533 B CN112370533 B CN 112370533B
- Authority
- CN
- China
- Prior art keywords
- bioluminescent
- fap
- block polymer
- probe
- imaging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000523 sample Substances 0.000 title claims abstract description 34
- 238000003384 imaging method Methods 0.000 title abstract description 18
- 229920000642 polymer Polymers 0.000 claims abstract description 12
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 8
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 230000004913 activation Effects 0.000 claims abstract description 4
- 210000004881 tumor cell Anatomy 0.000 claims abstract 2
- 238000001514 detection method Methods 0.000 claims description 16
- 108060001084 Luciferase Proteins 0.000 claims description 14
- 239000005089 Luciferase Substances 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 239000000693 micelle Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- JJTUDXZGHPGLLC-IMJSIDKUSA-N 4511-42-6 Chemical compound C[C@@H]1OC(=O)[C@H](C)OC1=O JJTUDXZGHPGLLC-IMJSIDKUSA-N 0.000 claims description 5
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 claims description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 229910021589 Copper(I) bromide Inorganic materials 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- UKODFQOELJFMII-UHFFFAOYSA-N pentamethyldiethylenetriamine Chemical compound CN(C)CCN(C)CCN(C)C UKODFQOELJFMII-UHFFFAOYSA-N 0.000 claims description 2
- 238000007872 degassing Methods 0.000 claims 2
- 239000008367 deionised water Substances 0.000 claims 2
- 229910021641 deionized water Inorganic materials 0.000 claims 2
- 238000003756 stirring Methods 0.000 claims 2
- 239000002202 Polyethylene glycol Substances 0.000 claims 1
- 238000006555 catalytic reaction Methods 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 238000012650 click reaction Methods 0.000 claims 1
- 230000002209 hydrophobic effect Effects 0.000 claims 1
- 239000003960 organic solvent Substances 0.000 claims 1
- 229920000747 poly(lactic acid) Polymers 0.000 claims 1
- 229920001223 polyethylene glycol Polymers 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 238000007789 sealing Methods 0.000 claims 1
- 238000005415 bioluminescence Methods 0.000 abstract description 22
- 230000029918 bioluminescence Effects 0.000 abstract description 22
- 210000004027 cell Anatomy 0.000 abstract description 17
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000012404 In vitro experiment Methods 0.000 abstract 1
- 229920001477 hydrophilic polymer Polymers 0.000 abstract 1
- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 abstract 1
- 230000007774 longterm Effects 0.000 abstract 1
- 150000003384 small molecules Chemical class 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 4
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 4
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 4
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 229920001400 block copolymer Polymers 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- -1 fluorescein amide Chemical class 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000004365 Protease Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000003999 initiator Substances 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 1
- 101710151806 72 kDa type IV collagenase Proteins 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 206010063560 Excessive granulation tissue Diseases 0.000 description 1
- 101150027576 FAP gene Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 206010006007 bone sarcoma Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000001126 granulation tissue Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000004204 optical analysis method Methods 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007151 ring opening polymerisation reaction Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
- A61K49/0082—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion micelle, e.g. phospholipidic micelle and polymeric micelle
-
- 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
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
- A61K49/0043—Fluorescein, used in vivo
-
- 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
Abstract
The invention provides a bioluminescent nanoprobe for long-term imaging of Fibroblast Activation Protein (FAP) in an organism. The bioluminescent nano probe comprises an amphiphilic block polymer and fluorescein loaded with the amphiphilic block polymer and having a bioluminescent function. The block polymer comprises: a hydrophilic polymer end, a lipophilic lactide end and an active peptide chain as a connecting region. The FAP-responsive bioluminescent nano probe provided by the invention has the advantages of long imaging time, high sensitivity, good selectivity and biocompatibility and the like. The invention also provides a preparation method and application of the probe. In vitro experiment results prove that the bioluminescence intensity and the FAP concentration present a good linear relationship, and the probe can be used for quantitatively detecting FAP. Meanwhile, compared with the traditional organic small-molecule bioluminescent probe, the nano probe is not easy to degrade and discharge by organisms, and long-time bioluminescent imaging of cell endogenous FAP is realized. In conclusion, the bioluminescent nano probe prepared by the invention is an effective tool for quantitatively detecting and imaging FAP in tumor cells for a long time.
Description
Technical Field
The invention belongs to the field of organic synthesis and detection, and particularly relates to a bioluminescent nano probe for detecting fibroblast activation protein, and a preparation method and application thereof.
Background
Bioluminescence imaging is a new technique for optical imaging using photons generated by enzymatic reactions in the body of an organism. The most common bioluminescent system is the firefly luciferase-luciferin system, the essence of which is that firefly luciferase catalyzes the substrate luciferin in the presence of energy (ATP) and oxygen, resulting in an enzymatic reaction to generate a bioluminescent signal.
Fibroblast Activation Protein (FAP) is a protein encoded by the FAP gene and having a molecular weight of 170kDa, and belongs to the homodimer. It is selectively expressed in epithelial cancer fibroblast response matrix, granulation tissue that heals wounds, and malignant cells of bone and soft tissue sarcomas. The protein is related to proliferation and growth of fibroblasts, tissue repair, epithelial-mesenchymal interaction, epithelial canceration and the like, is one of important markers of fibroblasts, has properties similar to other endopeptidases, and can preferentially cut Pro-Ala (proline-alanine) sequence peptide chains.
Bioluminescence imaging and fluorescence imaging are both common optical analysis methods used in chemical analysis. However, the current research shows that the fluorescence imaging needs additional exciting light, thereby causing the interference of the self-background fluorescence of the organism. Meanwhile, fluorescence imaging is also easily affected by external environments such as temperature, pH and other factors, and further the sensitivity of detection is affected. Bioluminescence imaging has its own distinct advantages over fluorescence imaging: (1) exogenous excitation light is not needed, so background signal interference does not exist, the luciferase catalytic oxidation luminescence efficiency is extremely high, the detection and the resolution are high, and the method is particularly suitable for high-sensitivity imaging under a complex biological background; (2) the tissue penetrating power is strong, and the device is more suitable for noninvasive, real-time and continuous in-vivo detection; (3) the substrate and enzyme involved in the bioluminescence are basically non-toxic to organisms and are safer. Based on the advantages of no autofluorescence interference, high signal-to-noise ratio, high sensitivity and the like of bioluminescence, the method is more suitable for in-vivo imaging application. According to previous literature reports, the half-life of D-fluorescein bioluminescence is short, typically less than 30 minutes. The fluorescein amide monomeric probes reported to date are also readily degradable, resulting in transient in vivo imaging of FAP. Therefore, it is promising to develop a method capable of monitoring imaging FAP for a long period of time.
Disclosure of Invention
The invention aims to provide a bioluminescent nanoprobe (PABC) capable of imaging FAP for a long time on the basis of the prior art, the probe can quantitatively detect FAP and provide cell bioluminescence imaging for 6 hours.
The invention also aims to provide a preparation method of the bioluminescent nanoprobe.
The invention also aims to provide the application of the bioluminescent nano probe in FAP detection.
The technical scheme of the invention is as follows:
the invention is based on the luciferase-luciferin bioluminescence imaging principle, takes a proline-alanine (Pro-Ala) sequence peptide chain as an FAP recognition group, takes luciferin as a luciferase recognition substrate, and constructs a bioluminescence nano Probe (PABC) for recognizing FAP.
The principle of the FAP recognition by the probe is as follows: when the fluorescein is coated by the amphiphilic polymer micelle, the fluorescein and external luciferase can not carry out enzymatic reaction, so that the PABC can not generate a bioluminescence signal; when the peptide chain of PABC is cut by FAP, the polymer particles are broken to release the luciferin entrapped inside, and then the luciferin is recognized by luciferase to perform an enzymatic reaction to generate bioluminescence. Is composed ofTo verify the reaction principle of the probe and FAP, PABC and FAP were reacted at 37 deg.C for 1 hr in phosphate buffer, and Mg was added 2+ (10mM), luciferase (10. mu.g/mL), and ATP (2mM) to generate a bioluminescent signal. Collecting bioluminescent signals through a small animal imager, and comparing intensity changes of the bioluminescent signals before and after response to realize qualitative and quantitative detection of FAP.
The detection method comprises the steps of adding FAP into a phosphate buffer solution of PABC, and reacting for 1 hour at 37 ℃. Then adding Mg 2+ A mixed solution of (10mM), luciferase (10. mu.g/mL) and ATP (2mM) was then rapidly placed in a small animal imager for bioluminescent signal acquisition.
The detection method is used in cell experiments. The nanoprobe solution was added to a 96-well plate incubated with luciferase-transfected MDA-MB-231 cells, and after 1 hour incubation, collection of bioluminescent signals was performed on a small animal imager.
Drawings
FIG. 1 shows PEG-N from top to bottom 3 Nuclear magnetic resonance hydrogen spectra of the intermediate product and the block copolymer;
FIG. 2 shows PEG-N from top to bottom 3 An infrared spectrum of the intermediate product and the block copolymer;
FIG. 3 is a transmission electron microscope image of the nanoprobe of the present invention before (A) and after (B) reaction with FAP;
FIG. 4 is a bioluminescence imaging graph of the in vitro response of the nanoprobe of the present invention to FAP of different concentrations;
FIG. 5 is a photo-graph of the selective detection of bioluminescence by the nanoprobe of the present invention;
FIG. 6 is a graph showing the results of cytotoxicity experiments using nanoprobes of the present invention;
FIG. 7 is a diagram of cellular bioluminescence imaging of the nanoprobe of the present invention. Blank control group (Blank), experimental group 1, experimental group 2 and reference group;
Detailed Description
The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.
Preparation of bioluminescent nanoprobes embodying the invention
The bioluminescent probe was prepared according to the following synthetic route:
(1) mixing PEG-N 3 (0.3g, 0.06mmol), alkynyl peptide GDRGETGPAC (60mg,0.06 mmol 1), DMF (3ml) and PMDETA (31mg, 0.18mmol) were charged to a Schlenk flask. The mixture was frozen in liquid nitrogen to a solid, degassed to vacuum and then back-filled with nitrogen. Under a nitrogen blanket, CuBr (26mg, 0.18mmol) was added. The system was degassed two more times by freeze-pump-thaw cycles and then sealed under vacuum. The reaction was carried out at 40 ℃ for 24 hours, and the mixture was then precipitated in cold diethyl ether solution. The crude product was collected by centrifugation and dried under vacuum overnight. The crude product was redissolved in water, added to a dialysis bag (MWCO:3000Da), and dialyzed against distilled water for 3 days. Freeze drying to obtain PEG-GDRGETGPAC-NH 2 And (3) solid powder. Yield: 52.1 percent.
(2) With PEG-GDRGETGPAC-NH 2 As an initiator, PEG-GDRGETGPAC-DLLA is synthesized by a ring-opening polymerization method. Firstly PEG-GDRGETGPAC-NH 2 The initiator being dissolved in CH 2 Cl 2 (0.5mL) was added with an excess of benzene and lyophilized to give an anhydrous white powder. Drying PEG-GDRGETGPAC-NH 2 (0.12 g,0.02mmol), D, L-lactide (0.288g, 2mmol), Sn (OCT) 2 (30. mu.l, 10mg/mL) and anhydrous THF (4mL) were added to a Schlenk flask and reacted at 80 ℃ for 18 hours. The reaction was precipitated by adding cold diethyl ether, and dried in a vacuum oven after repeating the cycle twice to obtain a white powdery block copolymer. Yield: 51.4 percent.
The beneficial effects of the invention are verified by experiments as follows:
the following experiments were used:
in vitro test for Probe Activity
Adding the nanoprobe (0.5Mg/mL) and FAP solution (specific concentration shown in figure 3) with different concentrations into a black 96-well plate, incubating at 37 ℃ for 30min, and adding Mg 2+ A mixed solution of (10mM), luciferase (10. mu.g/mL) and ATP (2mM) was prepared in 3 duplicate wells at each concentration, and then imaged under a small animal imager.
As a result, as shown in fig. 4, the bioluminescence intensity gradually increased with increasing FAP concentration. In vitro detection, the bioluminescence intensity and FAP show a good linear relation in a certain concentration range.
The results show that the probe has better detection sensitivity and can be used for quantitatively detecting trace FAP in a biological sample.
The invention takes a proline-alanine sequence peptide chain as a specific recognition site of FAP, designs an amphiphilic self-assembly polymer nano micelle, and prepares a bioluminescent nano probe for detecting FAP after loading fluorescein in the nano micelle. The bioluminescent nano probe is convenient and simple to prepare and high in yield. FAP can specifically recognize proline-alanine amido bond in the bioluminescent nanoprobe, so that a peptide chain is broken to release fluorescein in ATP, luciferase and Mg 2+ And the like, and the enzyme reaction is carried out to generate a bioluminescent signal. The bioluminescence signal intensity and the FAP concentration have a good linear relation in a certain range, and FAP can be quantitatively detected.
Selective detection experiment of Probe
In a black 96-well plate, nanoprobe solution (0.5mg/mL) and different analyte solutions (Blank, Glucose (10mM), DL-GSH (10mM), Esterase (20U/L), Catalase (20U/L), Hexokinase (20U/L), MMP-2(0.1mg/mL), Lysozyme (20U/L), Chromotprypsin (20U/L), Papain (10mM), Trypsin (0.1mg/mL), FAP (100ng/mL)) were added; incubating at 37 deg.C for 30min, adding Mg-containing solution to each well 2+ A mixed solution of (10mM), luciferase (10. mu.g/mL), and ATP (2mM) was imaged under a live imager.
The results are shown in fig. 5, and FAP produces strong bioluminescent signals, in contrast to the weak bioluminescent signals produced by a variety of active proteases and active molecules.
The experimental result shows that the probe can not be interfered by other substances, can specifically respond to FAP, and has good selectivity.
Luciferase-transfected MDA-MB-231 cytotoxicity assay for probes
By the MTT method (3- (4, 5-dimethylthiazole-2)-base) 2, 5-diphenyltetrazolium ammonium bromide (MTT) method to determine the cytotoxicity of probes against luciferase-transfected MDA-MB-231 cells. In 96-well plates, MDA-MB-231 cells were plated at 1X 10 per well 5 The cells were inoculated and cultured for 24 hours, and nanoprobe solutions (0, 0.05, 0.1, 0.2, 0.5, 0.8, 1.0mg/mL) were added at different concentrations, and five experiments were performed in parallel at each concentration. After the nanoprobes were incubated with the cells for 24 hours, MTT (10. mu.L) solution was added to each well and incubation was continued for 4 hours. The medium was removed and dimethyl sulfoxide (100 μ L) was added to completely dissolve the crystals, which were measured on a microplate reader and the absorbance at 570nm was recorded. The cellular activity calculation formula is as follows: cell viability (%) (mean of experimental group/mean of control group) × 100.
As shown in FIG. 6, the survival rate of the cells treated with the nanoprobes was over 85%, and the cell survival rate was high. The experimental result shows that the nano probe has good cell compatibility and can not kill cells. The interference of the change of the dosage of the compound on the bioluminescence signal of the cell in an in vivo experiment is eliminated.
Intracellular dynamic change detection experiment
Luciferase-transfected MDA-MB-231 cells were used for the experiment, and in a black 96-well plate, control 1 was incubated with the inhibitor SP-13786(100nM) for 1 hour with an equal amount of probe, control 2 was added with fluorescein (9. mu.M), blank control was added with PBS solution, and experimental was added with probe solution (0.5 mg/mL). Each group is provided with three multiple holes, after four groups of cells are respectively added with probe solution (0.5mg/mL), signal acquisition is carried out in a small animal imager, the time is recorded as 0min, then the change of bioluminescence intensity is recorded every 30min, and the total bioluminescence intensity in each hole is plotted.
The result is shown in fig. 7, no bioluminescent signal is generated in the blank group, and after the nano-probe is incubated in the experimental group, the luminescent signal can be detected in 30min, and the signal intensity reaches the maximum value in 120min and keeps a better luminescent intensity for a long time. In contrast, the control group incubated with FAP inhibitor earlier had a weaker signal. At the same time, the control group incubated with fluorescein alone, rapidly decayed after 30min reached its maximum signal intensity and disappeared completely at 90 min.
The experimental result shows that in a cell body, the nano probe can detect the endogenous FAP and can be used for long-time bioluminescence imaging in a living body.
In conclusion, the nano probe shows good selectivity and sensitivity in the aspect of target object detection, can be used for quantitative detection of FAP in cells, and has good application prospect. The results show that the imaging effect of the nano probe and FAP is good, and stable bioluminescent signals can be generated for a long time.
Claims (7)
1. A bioluminescent nanoprobe for detecting the content of Fibroblast Activation Protein (FAP) in an organism is an amphiphilic block polymer micelle of D-fluorescein wrapping a bioluminescent function, and the structure of the amphiphilic block polymer comprises: a hydrophilic polyethylene glycol terminus; a lipophilic polylactide end and a peptide chain which can be specifically destroyed by FAP, wherein the D-luciferin is positioned in a hydrophobic inner cavity of the micelle and separated from the luciferase to ensure that the nano probe is in an extinguishing state; the preparation method of the amphiphilic block polymer comprises the following steps:
2. the bioluminescent nanoprobe according to claim 1, wherein the preparation method of the amphiphilic block polymer comprises the following steps:
the first step is as follows: PEG-N 3 And peptidyl alkynyl GDRGETGPAC to make a first step product by Click reaction;
the second step is that: and (3) reacting the product obtained in the first step with D, L-lactide in an organic solvent to obtain the amphiphilic block polymer.
3. The bioluminescent nanoprobe according to claim 2, wherein in a first step, PEG-N 3 The molar ratio to peptidyl alkynyl GDRGETGPAC was 2: 1; then DMF and PMDETA were added together to a Schlenk flaskFreezing the mixture in liquid nitrogen to form solid, degassing to vacuum, and backfilling with nitrogen; adding CuBr for catalytic reaction under the protection of nitrogen, degassing the system for more than two times through a freezing-pump-unfreezing cycle, sealing under vacuum, reacting for 24 hours at 40 ℃, and precipitating the reaction residue in cold diethyl ether solution.
4. The bioluminescent nanoprobe of claim 2, wherein in a second step D, L-lactide is reacted with Sn (OCT) 2 And the first step product is reacted, wherein the molar ratio of the D, L-lactide to the first step product is 120: 1-50: 1; reacting at 80 ℃ for 24 hours under anhydrous and oxygen-free conditions.
5. The bioluminescent nanoprobe of claim 4, wherein the molar ratio of the D, L-lactide to the first step product is 100: 1.
6. The method of claim 1, wherein 1mg of the amphiphilic block polymer is added to 0.5mL of DMSO solution containing 1mg of D-fluorescein, 0.5mL of deionized water is slowly added under vigorous stirring, and after stirring at room temperature for 8 hours, the residue is dialyzed in deionized water for 24 hours using a dialysis bag to prepare micelles.
7. Use of the bioluminescent nanoprobe according to claim 1 for the preparation of a reagent for the detection of FAP in tumor cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010860240.4A CN112370533B (en) | 2020-08-24 | 2020-08-24 | Bioluminescent probe capable of imaging FAP for long time and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010860240.4A CN112370533B (en) | 2020-08-24 | 2020-08-24 | Bioluminescent probe capable of imaging FAP for long time and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112370533A CN112370533A (en) | 2021-02-19 |
| CN112370533B true CN112370533B (en) | 2022-09-13 |
Family
ID=74586652
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010860240.4A Active CN112370533B (en) | 2020-08-24 | 2020-08-24 | Bioluminescent probe capable of imaging FAP for long time and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112370533B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070087400A1 (en) * | 2004-07-30 | 2007-04-19 | Aldis Darzins | Covalent tethering of functional groups to proteins and substrates therefor |
| CN103012561A (en) * | 2012-12-26 | 2013-04-03 | 刘敏 | Small peptide for tumor targeted diagnosis |
| CN107648618A (en) * | 2017-09-27 | 2018-02-02 | 国家纳米科学中心 | A kind of drug delivery system and preparation method and application |
-
2020
- 2020-08-24 CN CN202010860240.4A patent/CN112370533B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070087400A1 (en) * | 2004-07-30 | 2007-04-19 | Aldis Darzins | Covalent tethering of functional groups to proteins and substrates therefor |
| CN103012561A (en) * | 2012-12-26 | 2013-04-03 | 刘敏 | Small peptide for tumor targeted diagnosis |
| CN107648618A (en) * | 2017-09-27 | 2018-02-02 | 国家纳米科学中心 | A kind of drug delivery system and preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| Noncovalently Caged Firefly Luciferins Enable Amplifiable Bioluminescence Sensing of Hyaluronidase‑1 Activity in Vivo;Jinqiu Luo et al.;《ACS Sens.》;20200522;第5卷;第1726-1733页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112370533A (en) | 2021-02-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Structural modification of BODIPY: Improve its applicability | |
| Zhao et al. | Fluorescent/phosphorescent dual-emissive conjugated polymer dots for hypoxia bioimaging | |
| Li et al. | Pathological‐condition‐driven construction of supramolecular nanoassemblies for bacterial infection detection | |
| US6201086B1 (en) | Electrically conductive electroactive functionalized conjugated polymers, and uses thereof | |
| JP2009106291A (en) | Method for rapidly detecting bacterial growth in culture | |
| EP3760193B1 (en) | Intracellular delivery vehicle | |
| CN104146964B (en) | Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof | |
| CN110982513A (en) | Preparation method of fluorescent carbon dots and application of fluorescent carbon dots in cell imaging | |
| CN115260083A (en) | Preparation method and application of mitochondrion-targeted viscosity response fluorescent probe | |
| EP3572800A1 (en) | Fluorescent gel for glucose detection and continuous glucose monitoring method using same | |
| Nan et al. | Chemiluminescence-triggered fluorophore release: Approach for in vivo fluorescence imaging of hydrogen peroxide | |
| Zhang et al. | Chemiluminescence chitosan hydrogels based on the luminol analog L-012 for highly sensitive detection of ROS | |
| CN112370533B (en) | Bioluminescent probe capable of imaging FAP for long time and application thereof | |
| CN108165267A (en) | A kind of switching mode pH fluorescence probes and its preparation method and application | |
| Lang et al. | A new poly (norbornene)-based sensor for fluorescent ratiometric sensing of adenosine 5′-triphosphate | |
| Huang et al. | Fabrication of claviform fluorescent polymeric nanomaterials containing disulfide bond through an efficient and facile four-component Ugi reaction | |
| Gong et al. | Polypeptide micelles with dual pH activatable dyes for sensing cells and cancer imaging | |
| CN113502159A (en) | Preparation method of pH activated carbon quantum dot emitting fluorescence in near-infrared region I, product and application thereof | |
| EP1152061B1 (en) | Method for screening physiologically active pyrrole imidazole derivative | |
| Geiselhart et al. | Untapped toolbox of luminol based polymers | |
| Liu et al. | Peroxynitrite-biosignal-responsive polymer micelles as intracellular hypersensitive nanoprobes | |
| CN109575240B (en) | Red light polymer with high fluorescence quantum efficiency, quantum dot solution and application | |
| CN113440612B (en) | Photodynamic enhanced polymer nanogel, preparation method and application thereof | |
| CN110642976B (en) | Polymer potassium ion fluorescent probe and preparation method and application thereof | |
| Hu et al. | Target-triggering, signal-amplified chemo/bio-sensors based on aggregation-induced emission luminogens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |