CN112370533A - 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
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- A61K49/00—Preparations for testing in vivo
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- 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
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- 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
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- 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
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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 technology for optical imaging using photons generated by enzymatic reactions in the body of a living being. The most common bioluminescent system is the firefly luciferase-luciferin system, which is essentially the enzyme luciferase catalyzes the substrate luciferin in the presence of energy (ATP) and oxygen to produce an enzymatic reaction that generates 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 expressed selectively in the response matrix of epithelial cancer fibroblasts, granulation tissue that heals the wound, 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 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, wherein 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. To verify the reaction principle of the probe with FAP, PABC were reacted with FAP in phosphate buffer at 37 ℃ for 1 hour, and Mg-containing solution was added2+(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 Mg2+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 bottom3Nuclear magnetic resonance hydrogen spectra of the intermediate product and the block copolymer;
FIG. 2 shows PEG-N from top to bottom3An 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 graphical representation of the selective detection of bioluminescence by the nanoprobes 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 graph 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 commercial products.
Preparation of bioluminescent nanoprobes embodying the invention
The bioluminescent probe was prepared according to the following synthetic route:
(1) mixing PEG-N3(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-NH2And (3) solid powder. Yield: 52.1 percent.
(2) With PEG-GDRGETGPAC-NH2As an initiator, PEG-GDRGETGPAC-DLLA is synthesized by a ring-opening polymerization method. Firstly PEG-GDRGETGPAC-NH2The initiator being dissolved in CH2Cl2(0.5mL) was added with an excess of benzene and lyophilized to give an anhydrous white powder. Drying PEG-GDRGETGPAC-NH2(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 deg.C for 30min, and adding Mg2+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 designs an amphiphilic self-assembly polymer nano micelle by taking a proline-alanine sequence peptide chain as a specific recognition site of FAP, 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 nano probe, so that a peptide chain is broken to release fluorescein in ATP, luciferase and Mg2+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, Gluco) were addedse (10mM), DL-GSH (10mM), Esterase (20U/L), Catalase (20U/L), Hexokinase (20U/L), MMP-2(0.1mg/mL), Lysozyme (20U/L), Chymotrypsin (20U/L), Papain (10mM), Trypsin (0.1mg/mL), FAP (100 ng/mL)); incubating at 37 deg.C for 30min, adding Mg-containing solution to each well2+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
Cytotoxicity of probes against luciferase-transfected MDA-MB-231 cells was determined by MTT method (3- (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium ammonium bromide (MTT) method MDA-MB-231 cells were plated at 1X 10 cells per well in 96-well plates5The 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 survival (%) (mean experimental/mean control) × 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 and an equal amount of probe was added, control 2 was added with fluorescein (9. mu.M), blank control was added with PBS solution, and experimental group 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 in advance 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 (8)
1. A bioluminescent nano probe for detecting the content of Fibroblast Activation Protein (FAP) in an organism is an amphiphilic block polymer micelle of fluorescein wrapping a bioluminescent function. The structure of the amphiphilic block polymer comprises: a hydrophilic polyethylene glycol terminus; lipophilic polylactide ends, and peptide chains that can be specifically disrupted by FAP. The luciferin is positioned in the hydrophobic inner cavity of the micelle and separated from the luciferase to ensure that the nano probe is in an extinguishing state.
2. When the bioluminescent nano probe reacts with FAP, the peptide chain part of the block polymer can be specifically cut by FAP, so that the polymer micelle is degraded. The polymer is degraded to release a large amount of loaded fluorescein, and then the fluorescein and luciferase in the system are subjected to enzymatic reaction to generate a bioluminescent signal, so that the bioluminescent analysis and detection of FAP are realized.
3. The method of preparing an amphiphilic block polymer according to claim 1, comprising the steps of:
4. a method for preparing an amphiphilic block polymer according to claim 3, characterized in that it comprises the following steps:
the first step is as follows: PEG-N3And peptidyl alkynyl GDRGETGPAC by Click reaction to make the first step product;
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.
5. The method for preparing bioluminescent probe according to claim 3, wherein in the first step, PEG-N3The molar ratio to peptidyl alkynyl GDRGETGPAC was 2: 1; DMF was then added to the Schlenk flask along with PMDETA. The mixture was frozen in liquid nitrogen to a solid, degassed to vacuum, and back-filled with nitrogen. Under the protection of nitrogen, CuBr is added for catalytic reaction. The system was degassed two more times by freeze-pump-thaw cycles, then sealed under vacuum, reacted at 40 ℃ for 24 hours and the reaction residue precipitated in cold diethyl ether solution.
6. The method of claim 3, wherein in the second step, D, L-lactide is mixed with Sn (OCT)2And a compound II, wherein the molar ratio of D, L-lactide to the compound II is 120: 1-50: 1; preferably 100: 1. Reacting at 80 ℃ under anhydrous and oxygen-free conditions for 24And (4) hours.
7. The method for preparing a bioluminescent probe micelle according to claim 1, wherein the amphiphilic block polymer (1mg) is added to a DMSO solution (0.5mL) containing D-fluorescein (1mg), and deionized water (0.5mL) is slowly added under vigorous stirring. After stirring at room temperature for 8h, the residue was dialyzed in deionized water for 24 hours with a dialysis bag to prepare micelles.
8. Use according to claim 2, characterized in that: the bioluminescent nano probe is applied to detecting FAP in tumor cells.
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| US20070087400A1 (en) * | 2004-07-30 | 2007-04-19 | Aldis Darzins | Covalent tethering of functional groups to proteins and substrates therefor |
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| 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 |
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