CN114806211A - Hydrophobic methylene blue fluorescent dye and preparation method and application thereof - Google Patents
Hydrophobic methylene blue fluorescent dye and preparation method and application thereof Download PDFInfo
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- methylene blue
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- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 229960000907 methylthioninium chloride Drugs 0.000 title claims abstract description 24
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 23
- 230000002209 hydrophobic effect Effects 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 238000002428 photodynamic therapy Methods 0.000 claims abstract description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 25
- NZYCYASKVWSANA-UHFFFAOYSA-M new methylene blue Chemical compound [Cl-].CCNC1=C(C)C=C2N=C(C=C(C(NCC)=C3)C)C3=[S+]C2=C1 NZYCYASKVWSANA-UHFFFAOYSA-M 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 11
- -1 perfluoro chain Chemical group 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000000799 fluorescence microscopy Methods 0.000 claims description 6
- 238000000967 suction filtration Methods 0.000 claims description 6
- BPHQIXJDBIHMLT-UHFFFAOYSA-N perfluorodecane Chemical compound FC(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F BPHQIXJDBIHMLT-UHFFFAOYSA-N 0.000 claims description 5
- 238000002390 rotary evaporation Methods 0.000 claims description 5
- 229920006395 saturated elastomer Polymers 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 abstract 1
- 229910052782 aluminium Inorganic materials 0.000 abstract 1
- 239000003504 photosensitizing agent Substances 0.000 description 10
- 238000010521 absorption reaction Methods 0.000 description 5
- ZKSVYBRJSMBDMV-UHFFFAOYSA-N 1,3-diphenyl-2-benzofuran Chemical compound C1=CC=CC=C1C1=C2C=CC=CC2=C(C=2C=CC=CC=2)O1 ZKSVYBRJSMBDMV-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
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- 239000000463 material Substances 0.000 description 3
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- OVBPIULPVIDEAO-LBPRGKRZSA-N Folic acid Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
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- 125000003929 folic acid group Chemical group 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
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- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- AMIBNSUSJRVQDL-UHFFFAOYSA-N 1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-docosafluorodecane hydroiodide Chemical compound I.FC(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F AMIBNSUSJRVQDL-UHFFFAOYSA-N 0.000 description 1
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- 230000017074 necrotic cell death Effects 0.000 description 1
- 125000005010 perfluoroalkyl group Chemical group 0.000 description 1
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- C09B21/00—Thiazine dyes
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Abstract
The invention discloses a hydrophobic methylene blue fluorescent dye and a preparation method and application thereof. The hydrophobic methylene blue fluorescent dye has a structural formula shown in a formula (I), can effectively release ROS to carry out photodynamic therapy or participate in subsequent reaction, can emit strong fluorescence at 650nm to carry out tumor imaging, has AIE (organic aluminum) property, can enhance luminescence, has strong hydrophobicity, can be conveniently encapsulated to form nanoparticles, and enhances the transportation and targeting functions.
Description
Technical Field
The invention relates to the field of material science, in particular to a hydrophobic methylene blue fluorescent dye and a preparation method and application thereof.
Background
Photodynamic therapy (PDT) is a treatment method based on interaction of light, photosensitizer and tissue oxygen, the action mechanism of the PDT is that the photosensitizer absorbs photons under illumination of specific wavelength, photosensitizer molecules are transited from a singlet state ground state (S0) to a singlet state excited state (S1) and finally reach a triplet excited state (T1), and collide with the tissue oxygen in the process of transiting from the triplet excited state back to the ground state to generate active oxygen (ROS), so that tumor cell necrosis and apoptosis are induced, and the purpose of cancer treatment is achieved. Compared with traditional cancer treatment means such as surgical resection, radiotherapy and chemotherapy, PDT has the advantages of non-invasiveness, rapid treatment process, good cell killing effect, repeated operation and the like, and becomes a tumor treatment method with development prospect.
A photosensitizer (photodynamic therapy drug), which is a special chemical substance different from a general chemotherapeutic drug, has a basic function of transferring energy, and it can absorb photons to be excited and rapidly transfer the absorbed light energy to another component molecule to be excited to generate a photochemical reaction, while the photosensitizer itself does not participate in the chemical reaction and returns to its original state (ground state). The photosensitizer is used as a core element of PDT, and mainly has the functions of absorbing light energy to generate ROS, and the cytotoxicity of ROS can make cells die so as to achieve the effect of eliminating tumors.
Methylene blue, a common dye-type photosensitizer, is commonly used in photodynamic antibacterial or antiviral therapy. The currently commonly used new methylene blue photosensitizer is water-soluble, is convenient to dissolve, but has poor biocompatibility in a mouse body, short half-life period and poor targeting property, and is difficult to enter cells.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a hydrophobic methylene blue fluorescent dye.
The hydrophobic methylene blue fluorescent dye has a structural formula shown as a formula (I):
wherein R is a saturated perfluoro chain and an isomer thereof. "+" indicates a positive charge.
Further, the saturated perfluoro chain and its isomer are selected from C 4 F 8 、C 5 F 11 、C 8 F 17 、C 9 F 19 、C 10 F 21 、C 11 F 23 、C 12 F 25 、C 15 F 31 Any one of them.
The invention also provides a preparation method of the hydrophobic methylene blue fluorescent dye, which comprises the following steps:
reacting new methylene blue with perfluoroalkyl at 80-150 ℃ for 24-36 hours to generate perfluorodecane new methylene blue, wherein the structural formula of the new methylene blue is shown as a formula (II),
the structural formula of the perfluorodecane methylene blue is shown as a formula (I).
Further, the method specifically comprises the following steps:
(1) charging new methylene blue and perfluoroalkyl iodide into a container, and then adding anhydrous organic solvent and 1-2 drops of triethylamine; filling nitrogen into the container;
(2) heating in an oil bath pan, and reacting in a dark place;
(3) performing suction filtration to remove redundant solids; carrying out rotary evaporation drying on the solution obtained by suction filtration, and continuously drying the dried solid in a vacuum drying oven;
(4) the dried solid is completely dissolved by water, then extracted and dried by rotary evaporation;
(5) and washing the dried solid with water to remove water-soluble impurities, collecting the residual solid, and drying in vacuum to obtain the hydrophobic methylene blue fluorescent dye.
Further, the mass ratio of the new methylene blue to the perfluoroalkane in the step (1) is 1: (1.2-3), wherein the mass ratio of the anhydrous organic solvent to the new methylene blue is (150-500): 1.
further, the anhydrous organic solvent is N, N-dimethylformamide or dimethyl sulfoxide.
Further, heating the oil bath kettle in the step (2) to 80-150 ℃, and reacting for 24-36h in a dark place at 300-800 rpm.
Further, the drying time of the vacuum drying oven in the step (3) is 24 to 36 hours.
Further, in the step (4), dichloromethane is used for extraction.
The invention also provides application of the hydrophobic methylene blue fluorescent dye in fluorescence imaging.
The invention also provides application of the hydrophobic methylene blue fluorescent dye in photodynamic therapy.
In summary, compared with the prior art, the invention achieves the following technical effects:
according to the invention, the hydrophobic side chain is connected to the new methylene blue to synthesize the NMBF, and the NMBF has strong hydrophobicity, so that the NMBF can be wrapped into nanoparticles to enhance the transportation and targeting functions, and meanwhile, tumor targeting groups such as folic acid and the like or other disease targeting groups can be coupled to the nanoparticles, thereby facilitating the construction of a treatment platform.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the synthesis of NMBF in accordance with the present invention;
FIG. 2 shows the solubility and luminescence properties of NMBF of the present invention in water and DMSO;
FIG. 3 is a diagram of the NMBF ROS release assay of the present invention;
FIG. 4 is a UV absorption curve of NMBF of the present invention;
FIG. 5 is a fluorescence emission image of NMBF of the present invention;
FIG. 6 is a fluorescence emission curve of NMBF of the present invention;
fig. 7 is a fluorescence imaging picture of macrophages using NMBF according to the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.
The new methylene blue and perfluoroalkyl iodide react for 24 to 36 hours at the temperature of between 80 and 150 ℃ to generate the new methylene blue derivative perfluorodecane new methylene blue NMBF of the invention, perfluoroalkyl iodide is adopted before the reaction, the reaction is a substitution reaction, and the compound after the reaction does not contain iodide ions. It can be used as AIE fluorescent dye for biological imaging, and can emit strong fluorescence at 650nm, or can utilize its AIE property to implement "Turn-on" and "Turn-off" of fluorescence. NMBF can also be used as photosensitizer for photodynamic therapy.
Example 1 design Synthesis of hydrophobic methylene blue fluorescent dye NMBF of the present invention
The method comprises the following specific steps:
(1) 40-70mg of New methylene blue and 80mg of perfluoroalkyl iodide were charged into a 50mL round bottom flask, followed by 8-10mL of DMF and 1-2 drops of triethylamine. The round bottom flask is connected with a three-way valve, and one end of the three-way valve is connected with a balloon filled with nitrogen.
The perfluoroalkyl iodide used in this example was perfluorodecane iodide, having the chemical formula:
when n is more than 2 and less than 5, the reaction temperature can be 50-80 ℃. When n > 5, the reaction temperature should be greater than 75 deg.C, and the reaction temperature should be gradually increased as n increases.
Because saturated perfluorinated chains and isomers thereof have similar structures and properties, one skilled in the art will appreciate that the side chain R group is C 4 F 8 、C 5 F 11 、C 8 F 17 、C 9 F 19 、C 10 F 21 、C 11 F 23 、C 12 F 25 、C 15 F 31 The technical effects of the present invention can be achieved.
(2) Heating to 80-90 ℃ in an oil bath kettle, 500rpm, and reacting for 24h in a dark place.
(3) And (5) carrying out suction filtration to remove redundant solid. And (4) carrying out rotary evaporation on the solution obtained by suction filtration, and drying the solid obtained after rotary drying in a vacuum drying oven for 24 hours.
(4) The dried solid was completely dissolved with water, followed by extraction with dichloromethane and spin-drying.
(5) And washing the dried solid with water to remove water-soluble impurities, collecting the residual solid, and drying in vacuum to obtain the product.
As shown in fig. 2, the prepared NMBF precipitated in water (shown in circles) and did not fluoresce because the dye quenched when insoluble, and the NMBF did not fluoresce in water, demonstrating its insolubility in water. While the luminescence in DMSO is strong, which proves that NMBF is dissolved in organic solvent.
Example 2 detection of ROS Release Using DPBF (1, 3-Diphenylisobenzofuran)
The method comprises the following specific steps:
(1) the uv spectrophotometer was calibrated to baseline with ultra pure water.
(2) 1-2mg of DPBF was dissolved in DMSO, then 8-10. mu.L of the solution was aspirated and added to 1mL of ultrapure water, mixed well and added to a 1mL cuvette, and the UV curve was measured.
(3) The cuvette was taken out, irradiated with a laser of 660nm power of 5W for 4-5min, and the UV curve was again tested.
(4) Repeating the step (3) for 3 times, and counting 5 ultraviolet curves.
(5) The values of each UV curve at 415nm were plotted in chronological order.
(6) The solution was reconstituted as in step (2) and DMSO-solubilized NMBF was added to a final concentration of 100-200mM/L and the final concentration of DPBF was maintained constant and loaded into a 1mL cuvette for UV measurement.
(7) And (5) repeating the steps (3), (4) and (5), and comparing the curve change before and after NMBF addition.
The results in fig. 3 illustrate that NMBF can release ROS under laser irradiation and is an excellent photosensitizer.
Example 3 ultraviolet spectrophotometer test NMBF ultraviolet absorption Curve and NMBF solution fluorescence Picture
The method comprises the following specific steps:
(1) NMBF was dissolved in DMSO at a concentration of 100-200mM/L and added to a 1mL cuvette.
(2) The ultraviolet absorption curve of the material in the range of 200-700nm is tested by an ultraviolet spectrometer.
(3) NMBF in DMSO at a concentration of 100-.
(4) The luminescence state of the NMBF solution at this time was photographed by a fluorescence imaging camera under irradiation with a 580nm laser.
Fig. 4 is an ultraviolet absorption curve of NMBF, and fig. 5 is a luminescence picture of NMBF solution under a fluorescence camera, indicating that it has luminescence properties.
Example 4 fluorescence Spectroscopy confirmed that NMBF has strong fluorescence at 650nm
The method comprises the following specific steps:
(1) NMBF was dissolved in DMSO at a concentration of 100-200mM/L and added to a 1mL cuvette.
(2) The absorption peak is tested by an ultraviolet spectrometer at 570-620 nm.
(3) The excitation at 570nm shows that the fluorescent material has stronger fluorescence at 650 nm.
FIG. 6 shows the fluorescence emission curve of NMBF, wherein the excitation wavelength of the fluorescence is 570nm and the emission peak of the fluorescence is 640 nm.
Example 5 fluorescence imaging of NMBF in cells
The method comprises the following specific steps:
(1) macrophages were cultured in 25mL flasks and passaged to 8-well confocal plates.
(2) NMBF was dissolved in DMSO at a concentration of 1-2 mg/mL.
(3) When the number of cells in the confocal culture plate is 3-5w, adding the solution in the step (2) into the confocal culture plate according to the volume ratio of 1:100, enabling the concentration of the solution in the culture medium to be 10-20 mu g/mL, and incubating for 20 min.
(4) Hoechst (fluorescent dye targeting the nucleus) staining was performed as described.
(5) The culture plate was imaged with a confocal microscope, and the channel was set to 580nm excitation for confocal imaging.
The results are shown in FIG. 7, where red fluorescence is NMBF fluorescence and blue fluorescence is Hoechst fluorescence, and the graphs are all shown in grayscale. Indicating that the NMBF of the present invention may be used for fluorescence imaging.
By combining the above embodiments, the invention discloses a hydrophobic methylene blue fluorescent dye, and a preparation method and application thereof. The hydrophobic methylene blue fluorescent dye has a structural formula shown in a formula (I), can effectively release ROS to carry out photodynamic therapy or participate in subsequent reaction, can emit strong fluorescence at 650nm to carry out tumor imaging, has AIE (advanced intermediate emission) property, can enhance luminescence, has strong hydrophobicity, can be conveniently encapsulated to form nanoparticles, enhances transportation and targeting functions, and can be coupled with tumor targeting groups such as folic acid and the like or other disease targeting groups on the nanoparticles to conveniently construct a therapy platform.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A hydrophobic methylene blue fluorescent dye, which is characterized in that the hydrophobic methylene blue fluorescent dye has a structural formula shown as a formula (I):
wherein R is a saturated perfluoro chain and an isomer thereof.
2. Hydrophobic methylene blue fluorescent dye according to claim 1, characterized in that the saturated perfluorinated chains and their isomers are selected from C 4 F 8 、C 5 F 11 、C 8 F 17 、C 9 F 19 、C 10 F 21 、C 11 F 23 、C 12 F 25 、C 15 F 31 Any one of them.
3. The method for preparing a hydrophobic methylene blue fluorescent dye according to claim 1, characterized by comprising the steps of:
reacting the new methylene blue with perfluoroalkyl iodide at the temperature of between 80 and 150 ℃ for 24 to 36 hours to generate perfluorodecane new methylene blue, wherein the structural formula of the new methylene blue is shown as a formula (II),
the structural formula of the perfluorodecane methylene blue is shown as a formula (I).
4. The preparation method according to claim 3, characterized by comprising the following steps:
(1) charging new methylene blue and perfluoroalkyl iodide into a container, and then adding anhydrous organic solvent and 1-2 drops of triethylamine; filling nitrogen into the container;
(2) heating in an oil bath pan, and reacting in a dark place;
(3) performing suction filtration to remove redundant solids; carrying out rotary evaporation drying on the solution obtained by suction filtration, and continuously drying the dried solid in a vacuum drying oven;
(4) the dried solid is completely dissolved by water, then extracted and dried by rotary evaporation;
(5) and washing the dried solid with water to remove water-soluble impurities, collecting the residual solid, and drying in vacuum to obtain the hydrophobic methylene blue fluorescent dye.
5. The production method according to claim 4, wherein the mass ratio of the new methylene blue to the perfluoroalkane in the step (1) is 1: (1.2-3), wherein the mass ratio of the anhydrous organic solvent to the new methylene blue is (150-500): 1.
6. the method according to claim 4, wherein the anhydrous organic solvent is N, N-dimethylformamide or dimethylsulfoxide.
7. The preparation method of claim 4, wherein the oil bath in step (2) is heated to 80-150 ℃ and 300-800 rpm, and is protected from light for 24-36 h.
8. The method according to claim 4, wherein the extraction in step (4) is carried out using methylene chloride.
9. Use of the hydrophobic methylene blue fluorescent dye according to any one of claims 1 to 2 in fluorescence imaging.
10. Use of a hydrophobic methylene blue fluorescent dye according to any one of claims 1 to 2 in photodynamic therapy.
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| CN114806211B (en) * | 2022-03-16 | 2023-04-07 | 深圳先进技术研究院 | Hydrophobic methylene blue fluorescent dye and preparation method and application thereof |
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