CN114525042B - Water-soluble croconic acid dye and preparation method and application thereof - Google Patents
Water-soluble croconic acid dye and preparation method and application thereof Download PDFInfo
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- CN114525042B CN114525042B CN202111611858.8A CN202111611858A CN114525042B CN 114525042 B CN114525042 B CN 114525042B CN 202111611858 A CN202111611858 A CN 202111611858A CN 114525042 B CN114525042 B CN 114525042B
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- croconic acid
- cyanine dye
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- dye
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000000980 acid dye Substances 0.000 title description 8
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims abstract description 44
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 28
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
- C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
- C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
- C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D333/30—Hetero atoms other than halogen
- C07D333/36—Nitrogen atoms
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a water-soluble croconic cyanine dye and a preparation method and application thereof, wherein the preparation method of the compound is simple, has obvious reaction sites and is easy to control the reaction process; croconic acid cyanine dye with good effectThe good molar absorptivity and ROS yield can be used for photodynamic therapy of diseases such as tumors and the like, and can also be used for pH response type photoacoustic imaging for tumor detection; has good fluorescence quantum yield, and can be used for pH response type fluorescence imaging of tumor detection.
Description
Field of the art
The invention belongs to the technical field of croconic acid cyanine dyes, and particularly relates to a water-soluble croconic acid cyanine dye, a preparation method thereof and application thereof in tumor photoacoustic imaging, fluorescence imaging and photodynamic therapy.
(II) background art
Accurate detection, diagnosis and excision of tumors depend on an accurate imaging system, and compared with traditional biological imaging technologies such as radiation imaging, ultrasonic imaging and magnetic resonance imaging, fluorescence imaging and photoacoustic imaging have great application potential in diagnosis and guided surgical excision due to the advantages of high sensitivity, high specificity, low cost, non-invasiveness, better biological safety and the like. Many fluorescent materials have also been widely used in fluorescence imaging studies, but most exhibit visible and near infrared first-region (NIR-I, 750-900 nm) emissions, which are attenuated by varying degrees of absorption and scattering upon entry into biological tissue, thereby reducing imaging depth and contrast. The near infrared two region (NIR-II) has the advantages of longer emission wavelength (1000-1700 nm), stronger penetrability of biological tissues, deeper detection depth, higher spatial resolution and the like, and becomes a hot spot direction of biomedical research. At present, some existing inorganic materials such as rare earth down-conversion nano particles, carbon nano tubes, quantum dots and the like can realize near infrared two-region emission, but most of emission wavelengths of the inorganic materials are positioned in a near infrared one region, and the defects of poor biological safety performance of heavy metals, slow metabolism after entering a living body and the like limit the application of the inorganic materials. Compared with inorganic materials, the organic croconic acid fluorescent dye has smaller relative molecular weight, is easy to metabolize, has better biocompatibility and stability, is easier to modify, and can realize the emission of the near infrared second window region.
The photoacoustic imaging technology is used as an emerging nondestructive and noninvasive composite imaging technology, has the characteristics of high sensitivity of optical imaging and high penetrability and resolution of acoustic imaging, and has wide application prospects in biomedical clinical diagnosis, especially in the aspects of in-vivo tissue structure and functional imaging. In order to overcome the scattering effect of light, improve the photoacoustic signal-to-noise ratio and enhance the imaging quality of the photoacoustic imaging technique, the use of an exogenous photoacoustic contrast agent is another effective method in addition to selecting an appropriate wavelength region as a working region. By using the exogenous near infrared contrast agent, the penetration depth of the photoacoustic imaging can be increased, the maximum allowable irradiation energy can be increased, the background signal can be reduced, the optical and acoustic properties of local tissues can be changed, and the contrast and resolution of the imaging can be increased, so that the imaging effect of the photoacoustic imaging can be obviously enhanced.
Photodynamic therapy is a new technology for diagnosing and treating diseases by utilizing photodynamic effect, and the process is that under the irradiation of laser with specific wavelength, photosensitizer distributed in tissue is excited, and the excited photosensitizer transfers energy to surrounding oxygen to generate singlet oxygen with strong activity, and the singlet oxygen and adjacent biological macromolecules undergo oxidation reaction to produce cytotoxicity, so that the cells are damaged or even die.
Therefore, the invention tries to develop the water-soluble croconic cyanine dye with absorption in the near infrared region, can realize the combination of photodynamic therapy, tumor microenvironment pH response type fluorescence imaging technology and photoacoustic imaging technology, can realize the complementary advantages among the imaging technologies, enhances fluorescence and photoacoustic response values in the acidic environment of tumor parts, provides more abundant biological information for disease diagnosis, simultaneously realizes the purpose of diagnosis and treatment, avoids risks and burdens caused by injecting different contrast agents for multiple times, and has wide application prospect.
(III) summary of the invention
The invention aims to provide a pH response type croconic acid cyanine dye with a photodynamic effect for the first time, a preparation method thereof and application thereof in preparing an optical imaging reagent or a tumor treatment medicament, wherein the croconic acid cyanine dye can enhance the photoacoustic and fluorescence imaging effects under the acidic microenvironment of tumors, shows tumor tissue boundaries under the excitation of near infrared light, provides a relatively objective reference for complete excision of tumors, and also reports that the croconic acid cyanine dye can be used for photodynamic treatment of tumors for the first time and effectively necrotizes tumor tissues.
The technical scheme adopted by the invention is as follows:
the invention provides a water-soluble croconic cyanine dye shown in a formula (I):
the invention also provides a preparation method of the croconic cyanine dye, which comprises the following steps:
(1) Synthesis of intermediates of formula B:
adding the compound A and N-methylpiperazine into toluene, uniformly mixing, heating to 110-120 ℃ for reflux reaction for 8 hours, and removing the solvent by rotary evaporation of the reaction solution to obtain oily concentrate; dissolving the oily concentrate with petroleum ether, performing silica gel chromatographic column with mixed solvent of petroleum ether and ethyl acetate at volume ratio of 4:1 as eluent, eluting at eluting speed of 1-5mL/min (preferably 1 mL/min) for 5-7 (preferably 5) column volumes to remove unreacted compound A; eluting 8-9 (preferably 9) column volumes with a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 1:1 as an eluent at an eluting speed of 1-5mL/min (preferably 3 mL/min), performing thin-layer chromatography monitoring with a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 4:1 as a developing agent, collecting components with Rf value of 0.1-0.3, and rotary evaporating to dryness to obtain an intermediate B;
(2) Synthesizing a target product shown in a formula (I):
dissolving intermediate B and croconic acid (4, 5-dihydroxy-4-cyclopentene-1, 2, 3-trione) in n-butanol and toluene, and heating to 110-120Reflux-reacting at deg.C overnight, rotary evaporating to dryness to remove solvent, dissolving the concentrate with mobile phase, and separating and purifying by preparative high performance liquid chromatography (chromatographic column model is COSMIL 5C) 18 MS-II 20mm 250 mm) in a volume ratio of 1:9 of ultrapure water: eluting with methanol mixed solvent as mobile phase at eluting speed of 2-6mL/min (preferably 4 mL/min), detecting wavelength of 700nm, column temperature of 30deg.C, collecting eluate with retention time of 14min, rotary evaporating to dryness of methanol, lyophilizing (preferably 4 deg.C), and obtaining croconic cyanine dye shown in formula (I), and recording as SR780.
Further, the mass ratio of the compound A to the N-methylpiperazine in the step (1) is 1:1-5, preferably 1:1.3; the toluene volume is used in an amount of 20 to 50mL/g, preferably 32mL/g, based on the mass of compound A.
Further, in step (2), the mass ratio of intermediate B to croconic acid is 1:0.3-0.5, preferably 1:0.32; the volume usage of the n-butanol is 100-200mL/g, preferably 148mL/g, based on the mass of the intermediate B; the volume ratio of the n-butanol to the toluene is 1:1.
the invention also provides application of the croconic acid cyanine dye shown in the formula (I) in preparation of tumor imaging reagents, and the croconic acid cyanine dye is used for photoacoustic imaging or fluorescence imaging of tumor detection.
The invention also provides application of the croconic acid cyanine dye shown in the formula (I) in preparing tumor cell activity inhibitors, wherein the tumor cells comprise breast cancer cells, oral cancer cells, liver cancer cells, lung cancer cells, stomach cancer cells, pancreas cancer cells, colorectal cancer cells, bladder cancer cells, prostate cancer cells and other solid tumor cells. More specifically, the liver cancer cells include HepG-2, 7404 and 4T1 of breast cancer cells.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a symmetrical croconic cyanine dye SR780, which expands the pi conjugation degree of a system by introducing electron donating groups and has a donor-acceptor-donor structure to form a rigid resonance plane structure for stabilizing zwitterionic ions, so that the absorption and emission wavelength of the cyanine dye can be effectively regulated and controlled, and the cyanine dye can be red-shifted to a near infrared region (750-1000 nm) with longer wavelength. The compound has the advantages of simple preparation method, obvious reaction sites and easy control of reaction progress.
The croconic acid cyanine dye SR780 provided by the invention has good absorption performance in a near infrared region, and can be used for photoacoustic imaging of tumor detection.
The croconic acid cyanine dye SR780 provided by the invention has good fluorescence quantum yield of 2.058% (which is obtained by using a 900LP filter by using a carbon nano tube as a contrast and 808nm excitation) and can be used for fluorescence imaging of tumor detection.
The croconic acid cyanine dye SR780 provided by the invention has good ROS production efficiency, and can be used for photodynamic therapy of tumor therapy.
(IV) description of the drawings
FIG. 1 is a nuclear magnetic resonance spectrum of the croconic acid cyanine dye SR780 prepared in example 2.
Fig. 2 is a nuclear magnetic carbon spectrum of the croconic acid cyanine dye SR780 prepared in example 2.
Fig. 3 is an ultraviolet-visible absorption spectrum of the croconic acid cyanine dye SR780 in example 3.
FIG. 4 is a fluorescence emission spectrum of the croconic acid cyanine dye SR780 in example 3.
FIG. 5 is a graph showing fluorescence emission patterns of the croconic acid cyanine dye SR780 of example 3 under different pH conditions.
Fig. 6 is a photoacoustic signal spectrum of the croconic acid cyanine dye SR780 in example 3.
Fig. 7 shows the uv-vis absorption spectrum of the croconic acid cyanine dye SR780 of example 4 after laser irradiation for various times.
FIG. 8 is an ultraviolet-visible absorption spectrum of a commercially available dye ICG of example 4 after laser irradiation for various times.
FIG. 9 is an ultraviolet-visible absorption spectrum of a commercially available dye IR780 laser irradiation of example 4 after various times.
Fig. 10 is a fluorescence emission spectrum of the croconic acid cyanine dye SR780 in example 5 at different irradiation times.
FIG. 11 is a plot of ROS yield of the croconic acid cyanine dye SR780 in example 5.
FIG. 12 is a graph showing the effect of the ROS productivity of croconic acid dye 1 in example 5.
FIG. 13 is a graph showing the effect of ROS yield on the commercially available dye ICG of example 5.
FIG. 14 is a bar graph showing the effect of croconic acid cyanine dye concentration on survival of hepatoma cells HepG-2 in example 6.
FIG. 15 is a bar graph showing the effect of ICG concentration of commercially available dye on survival of hepatoma cells HepG-2 in example 6.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the room temperature of the invention is 25-30 ℃.
Example 1 Synthesis of intermediate B
Compound A (0.263 g) and N-methylpiperazine (0.341 g) were charged into a round-bottomed flask containing 8ml of toluene, heated to 110-120℃and subjected to reflux reaction for 8 hours, and the solvent was removed by rotary evaporation of the reaction solution to obtain an oily concentrate. The oily concentrate was dissolved in 10mL of petroleum ether and used as a loading solution for silica gel column chromatography.
Filling 50g of 100-200 mesh column chromatography silica gel and 50mL of petroleum ether into a chromatographic column (3 cm multiplied by 30 cm), slowly loading 10mL of loading liquid along the tube wall after releasing petroleum ether, and mixing petroleum ether with the volume ratio of 4:1: the mixed solvent of ethyl acetate is used as an eluent, and after 6 column volumes are eluted at an elution speed of 1mL/min to remove the compound A, petroleum ether with a volume ratio of 1:1 is used instead: eluting with mixed solvent of ethyl acetate, eluting with 9 column volumes at an elution rate of 3mL/min, performing thin layer chromatography monitoring with mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 4:1 as developing agent, collecting components with Rf value of 0.1-0.3, and vacuum rotary evaporating the mixed solvent to obtain compound B0.298 g.
Nuclear magnetic data of compound B: 1 H NMR(400MHz,DMSO)δ6.80(t,J＝4.0Hz,1H),6.63(d,J＝8.0Hz,1H),7.74(d,J＝4Hz,1H),3.20(t,J＝4Hz,4H),2.58(t,J＝4Hz,4H),2.36(s,3H). 13 C NMR(101MHz,DMSO)δ159.24,126.68,112.29,105.31,54.39,51.37,46.11.
example 2 synthesis of croconic acid cyanine dye SR780.
Intermediate B (0.1 g) prepared in the method of example 1 and croconic acid (0.031 g) were charged into a round bottom flask containing n-butanol (15 mL) and toluene (15 mL), and heated under reflux at 110 ℃ for reaction overnight, and the reaction solution was rotary evaporated to dryness to remove the solvent, thereby obtaining a solid concentrate. The solid concentrate was treated with 20mL of ultrapure water in a volume ratio of 1:9: and (3) after dissolving the methanol mixed solvent, separating and purifying by adopting a preparative high performance liquid chromatograph, collecting and obtaining eluent when the retention time is 14min according to chromatographic peak signals, rotationally evaporating the methanol, and freeze-drying at 4 ℃ to obtain 0.062g of croconic acid cyanine dye (marked as SR 780) shown in the formula (I). The nuclear magnetic hydrogen spectrum is shown in figure 1, and the nuclear magnetic carbon spectrum is shown in figure 2.
The model of the prepared high performance liquid chromatograph is LC3000, and the model of the chromatographic column is COSMIL 5C18-MS-II 20mm 250mm; the mobile phase is ultrapure water with the volume ratio of 1:9: a methanol mixed solvent; the elution rate was 4mL/min, the detection wavelength was 700nm, the column temperature was 30℃and the retention time was 14min.
Nuclear magnetic data of SR 780: 1 H NMR(600MHz,DMSO)δ8.54(s,2H),7.04(s,2H),3.72(s,3H),2.51(s,8H),2.26(s,8H),1.23(s,3H). 13 C NMR(151MHz,DMSO)δ181.94,173.38,123.16,115.00,70.25,69.76,65.96,63.10,54.06,51.22,45.66,31.76,29.35,28.93,24.92,22.56,14.43.
example 3, absorption Spectrum and emission Spectrum detection of croconic acid cyanine dye
Ultraviolet visible absorption spectrum test: ultraviolet-visible absorption spectrum detection was performed on the croconic acid cyanine dye SR780 prepared by the method of example 2 using an ultraviolet-visible spectrophotometer (UV-5500 PC, shanghai meta-analysis instruments limited): 700. Mu.L of SR780 solution with the concentration of 7.5. Mu.g/mL (the SR780 is firstly prepared into 1mM mother solution by using dimethyl sulfoxide (DMSO), then the mother solution is diluted to 7.5. Mu.g/mL by using deionized water), the mother solution is placed in a quartz cuvette, the deionized water is used for setting to zero, the wavelength range of 500 nm-1000 nm is selected for spectral scanning, and the result is shown in figure 3, and the SR780 has strong absorption between 600nm and 900 nm.
Fluorescence emission spectrum test: fluorescence emission spectroscopy detection of the croconic acid cyanine dye SR780 prepared by the method of example 2 was performed using a fluorescence spectrophotometer (PerkinElmer Lambda 750): 3mL of SR780 solution with the concentration of 10 mu M (the SR780 is firstly prepared into 1mM mother solution by using dimethyl sulfoxide (DMSO), then the mother solution is diluted to 10 mu M by using deionized water), and the wavelength range of 900 nm-1400 nm is selected for spectral scanning, and the result is shown in FIG. 4, the SR780 has wide emission between 900nm and 1100 nm.
In addition, 3mL of SR780 solution (SR 780 was first prepared as a 1mM stock solution using dimethyl sulfoxide DMSO and then diluted to 10. Mu.M with deionized water) at a concentration of 10. Mu.M and at a pH of 1.0, 3.0, 4.5, 5.5, 6.5, 7.4, 8.0, respectively, was prepared, and the spectral scan was performed by selecting a wavelength range of 900nm to 1400nm, as shown in FIG. 5, the fluorescence signal of SR780 was gradually increased as the pH was decreased between 900nm and 1100 nm.
Photoacoustic signal testing: photoacoustic signal detection was performed on the croconic dye SR780 prepared in example 2 using a photoacoustic imager (Vevo LAZR, FUJIFILM visual sonic): 200 mu L of SR780 DMSO solution with the concentration of 5mM is taken, excitation light of 1200 nm-2000 nm is selected, and the result is shown in FIG. 6, and SR780 has strong sound signals at 1400 nm-1800 nm.
The result shows that the absorption spectrum and the emission spectrum of the SR780 are both in the near infrared region (750-900 nm), the emission spectrum extends to the near infrared two regions (1000-1100 nm), and the SR has stronger fluorescence and photoacoustic signals.
Example 4 stability of croconine dye compared with commercially available dye ICG and IR780
The croconic acid cyanine dye SR780, indocyanine green (ICG, TCI) and IR780 iodide (HEOWNS) prepared in the method of example 2 were dissolved in DMSO to prepare 1mM mother solutions, and each was diluted with deionized water to prepare a solution having a concentration of 10. Mu.g/mL. 1mL each was placed in an EP tube, and a 808nm laser (power 1.5W/cm) 2 ) Respectively irradiating for 0min, 10min and 20min, and selecting wavelength range of 500-1000 nm for ultraviolet-visible absorption spectrum scanning to obtain the final productAs shown in fig. 7, 8 and 9, the commercially available dye ICG and IR780 have no ultraviolet absorption after 10min of illumination, while the croconic dye SR780 has almost no change in ultraviolet absorption after 20min of illumination, which indicates that the SR780 has good stability.
Example 5 ROS productivity Effect of croconic dye SR780
1. DCFH solution:
mu.L of 10mM active oxygen fluorescent probe (DCFH-DA) methanol solution and 20 mu.L of 0.01M NaOH aqueous solution are taken and reacted for 30min at room temperature, 975 mu.L of PBS is added, and 50 mu.M of non-fluorescent 2',7' -Dichlorofluorescein (DCFH) solution is obtained.
2. Influence of laser irradiation time on dye fluorescence intensity
The gram-ketocyanine dye SR780 prepared in example 2 was dissolved in deionized water to prepare an SR780 solution having a concentration of 5 μm. To 990. Mu.L of SR780 solution, 10. Mu.L of the DCFH (50. Mu.M) solution prepared in step 1 was added to form a 1mL reaction system, and the DCFH working concentration in the reaction system was maintained at 0.5. Mu.M. With 808nm laser (power 0.5W/cm) 2 ) The irradiation was continued for 180s, and fluorescence was detected in the wavelength range of 450-650nm at the irradiation of 0, 10, 20, 30, 45, 60, 90, 120 and 180s, respectively. As a result, as shown in FIG. 10, the fluorescence at a wavelength of 525nm was gradually increased with the lapse of time.
3. Influence of laser irradiation time on dye fluorescence intensity
A Blank group (Blank), an SR780 non-illumination group (SR 780), an SR780 illumination group (SR 780+Laser), an ICG illumination group (ICG+Laser) and an SR780 and vitamin C illumination group (SR 780+VC+Laser) are arranged.
SR780 illumination group: to 990. Mu.L of 5. Mu.M SR780 solution (1 mM stock solution prepared by preparing SR780 from dimethyl sulfoxide DMSO and diluting the stock solution to 5. Mu.M with deionized water) was added 10. Mu.L of the DCFH (50. Mu.M) solution prepared in step 1 to construct a 1mL reaction system, and the DCFH working concentration in the reaction system was maintained at 0.5. Mu.M. With 808nm laser (power 0.5W/cm) 2 ) The irradiation was continued for 180s.
SR780 no illumination group: to 990. Mu.L of 5. Mu.M SR780 solution (1 mM stock solution prepared by preparing SR780 from dimethyl sulfoxide DMSO and diluting the stock solution to 5. Mu.M with deionized water) was added 10. Mu.L of the DCFH (50. Mu.M) solution prepared in step 1 to construct a 1mL reaction system, and the DCFH working concentration in the reaction system was maintained at 0.5. Mu.M. The mixture was left in the dark for 180s.
SR780 plus vitamin C light group: to 990. Mu.L of 5. Mu.M SR780 solution (1 mM mother liquor prepared by preparing SR780 with dimethyl sulfoxide DMSO and diluting the mother liquor to 5. Mu.M with deionized water) 10. Mu.L of the DCFH (50. Mu.M) solution prepared in step 1 and 88. Mu.g of vitamin C were added to form a 1mL reaction system, and the working concentration of DCFH in the reaction system was maintained at 0.5. Mu.M. With 808nm laser (power 0.5W/cm) 2 ) The irradiation was continued for 180s.
ICG illumination group: to 990. Mu.L of a 5. Mu.M ICG solution (1 mM mother liquor prepared by preparing ICG with dimethyl sulfoxide DMSO and diluting the mother liquor to 5. Mu.M with deionized water) was added 10. Mu.L of the DCFH (50. Mu.M) solution prepared in step 1 to construct a 1mL reaction system, and the DCFH working concentration in the reaction system was maintained at 0.5. Mu.M. With 808nm laser (power 0.5W/cm) 2 ) The irradiation was continued for 180s.
Blank control group: 10. Mu.L of the DCFH (50. Mu.M) solution prepared in step 1 was added to 990. Mu.L of water to construct a 1mL reaction system, and the DCFH working concentration in the reaction system was maintained at 0.5. Mu.M. With 808nm laser (power 0.5W/cm) 2 ) The irradiation was continued for 180s.
Fluorescence intensity at 525nm wavelength was detected by a fluorescence spectrophotometer at each of the groups at laser irradiation of 0, 10, 20, 30, 45, 60, 90, 120 and 180s, respectively, and the ratio of fluorescence intensity at each time point to fluorescence intensity at 0s (i.e., reactive oxygen species ROS yield) is shown in FIG. 11, SR780 shows higher ROS yield compared to the commercially available dye ICG group having similar ultraviolet absorption.
4. Photodynamic forces of different dyes
Reference [1]Spence G T,Hartland G V,Smith B D.Activated photothermal heating using croconaine dyes[J ] Chemical Science 2013,4 (11): 4240 croconic acid dye 1 was prepared.
To 2mL of a 5. Mu.M methanolic solution of croconic acid dye 1 was added 20. Mu.L of a 3-Diphenylisobenzofuran (DPBF) chloroform solution (10 mM) to constitute a 2.02mL reaction system, and the working concentration of DPBF in the reaction system was 0.1mM. The irradiation was continued for 180s with a xenon lamp (power 150W,620nm filter) at a distance of 15 cm. Samples were taken at 0, 1,2, and 3min, and scanned by an ultraviolet spectrophotometer at 300-950nm, and the results are shown in FIG. 12.
Under the same conditions, croconic acid dye 1 was replaced with commercially available dye ICG, and the results are shown in fig. 13.
Fig. 12, 13 show that ICG group gradually decreased in absorbance at 400nm with prolonged illumination time, indicating ROS production, whereas croconic acid dye 1 group did not change in absorbance at 400nm with prolonged illumination time, indicating no ROS production. From this result, it was found that SR780 has a much higher photodynamic effect (fig. 10 and 11) than the croconic acid dye 1 in the literature, that SR780 generates more ROS after being irradiated with light (fig. 10 and 11), and that the effect is superior to ICG.
EXAMPLE 6 anti-HepG-2 tumor cell proliferation Activity of croconic dye SR780
1. This example demonstrates the photodynamic therapeutic effect of croconic dye on tumor cells.
(1) The croconic acid cyanine dye SR780 prepared in the method of example 2 was dissolved in PBS to prepare a 1mM stock solution, and diluted with PBS and administered with a concentration gradient (10 μm, 20 μm, 40 μm, 60 μm, 80 μm) respectively.
(2) The metabolic activity of SR780 on liver cancer cells HepG-2 was evaluated by MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide) method, and the specific steps are as follows:
inoculating liver cancer cell HepG-2 into DMEM complete medium containing 10% fetal bovine serum, placing into incubator (37deg.C, 5% CO) 2 ) After incubation for 3 passages, cells in logarithmic phase were inoculated in 96-well plates at a density of 8×10 3 Cells/wells were incubated overnight at 37 ℃ to allow the cells to adhere well. Old medium was aspirated and experimental and control groups were set, the experimental groups being light (sr780+l) and non-light (SR 780), each group being set with 4 replicates (calculation error).100. Mu.L of serum-free DMEM medium containing SR780 at various concentrations (10. Mu.M, 20. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M) was added to each of the light (SR 780+L) and non-light (SR 780), and 100. Mu.L of serum-free DMEM medium was added to the blank. Continuing with 37℃and 5% CO 2 After 4h of incubation, the light group was given 0.5W/cm 2 For 2min, the non-illuminated group and the blank group were dark treated. After the incubation is continued for 24 hours after the irradiation, the absorbance of each group at 490nm is detected by an enzyme-labeled instrument (Thermo ScientificTMMultiskanTMFC), and the influence of the drug on the survival rate of tumor cells is calculated by the ratio of the absorbance of the experimental group to that of the blank control group. The results are shown in FIG. 14. From the results, it can be seen that the croconic cyanine dyes show dose-dependent photo cytotoxicity, and the cell viability decreases with increasing dye concentration under light conditions, indicating that they have high cell killing efficiency under these conditions. At the same time, these dyes were found to have high cell viability in the dark, indicating that they are non-toxic in the absence of light.
2. Photodynamic therapy effect of commercial dye ICG on tumor cells
(1) Commercial dye ICG was dissolved in DMSO to prepare 100mM stock solutions, which were diluted with PBS and administered with a concentration gradient of (0. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, 250. Mu.M) respectively.
(2) Inoculating liver cancer cell HepG-2 into DMEM complete medium containing 10% fetal bovine serum, placing into incubator (37deg.C, 5% CO) 2 ) After incubation for 3 passages, cells in logarithmic phase were inoculated in 96-well plates at a density of 8×10 3 Cells/wells were incubated overnight at 37 ℃ to allow the cells to adhere well. Old medium was aspirated and experimental and control groups were set, the experimental groups being light and non-light groups, each group being set with 4 replicates (calculation errors). 100. Mu.L of DMEM medium containing different concentrations (0. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, 250. Mu.M) of ICG was added to each of the light group (ICG+L) and the non-light group (ICG), and 100. Mu.L of DMEM medium was added to the blank group. Continuing with 37℃and 5% CO 2 After 4h of incubation, the light group was given 0.2W/cm 2 Is irradiated for 2min with 810nm laser, and dark treatment is performed on the non-light group and the blank control group. Subsequent to irradiationAfter incubation for 24 hours, the absorbance of each group at 490nm is detected by an ELISA detector, and the influence of the drug on the cell survival rate is calculated by the ratio of the absorbance of the experimental group to that of the blank control group. The results are shown in FIG. 15. From the results, it can be seen that the cell viability of ICG decreases with increasing dye concentration under light conditions (ICG killer cell action includes both aspects, i.e. photodynamic and photothermal), but the concentration of ICG required is much higher than SR780 when compared to SR780 which only produces photodynamic effects, indicating that the photodynamic effects of SR780 alone are better than the combined effects of ICG photodynamic and photothermal.
The results show the application prospect of the croconic acid cyanine dye in photodynamic therapy.
Claims (9)
1. A water-soluble croconic cyanine dye of formula (I):
2. a process for the preparation of a croconic acid cyanine dye according to claim 1, characterized in that the process comprises the steps of:
(1) Synthesis of intermediates of formula B:
adding the compound A and N-methylpiperazine into toluene, uniformly mixing, heating to 110-120 ℃ for reflux reaction for 8 hours, and removing the solvent by rotary evaporation of the reaction solution to obtain oily concentrate; dissolving the concentrate with petroleum ether, performing silica gel column chromatography, eluting with mixed solvent of petroleum ether and ethyl acetate at volume ratio of 4:1 as eluent, and eluting at speed of 1-5mL/min for 5-7 column volumes; eluting with mixed solvent of petroleum ether and ethyl acetate at volume ratio of 1:1, and eluting with 1-5mL/min for 8-9 column volumes; carrying out thin layer chromatography monitoring by taking a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 4:1 as a developing agent, collecting components with Rf value of 0.1-0.3, and carrying out rotary evaporation to dryness to obtain an intermediate B;
(2) Synthesizing a target product shown in a formula (I):
dissolving an intermediate B and croconic acid in n-butyl alcohol and toluene, heating to 110-120 ℃ for reflux reaction overnight, rotationally evaporating the reaction solution until the solvent is removed, dissolving the obtained concentrate by using a mobile phase, separating and purifying by adopting a preparative liquid chromatograph, collecting an eluent with retention time of 14min, rotationally evaporating the methanol, and freeze-drying to obtain the croconic acid cyanine dye shown in the formula (I); the mobile phase is a mixed solvent of ultrapure water and methanol in a volume ratio of 1:9; the elution speed of the mobile phase is 2-6mL/min, the detection wavelength is 700nm, and the column temperature is 30 ℃.
3. The method of claim 2, wherein the mass ratio of compound a to N-methylpiperazine in step (1) is 1:1-5; the volume amount of toluene is 20-50mL/g based on the mass of the compound A.
4. The process of claim 2, wherein in step (2), the mass ratio of intermediate B to croconic acid is 1:0.3-0.5; the volume dosage of the n-butanol is 100-200mL/g based on the mass of the intermediate B; the volume ratio of the n-butanol to the toluene is 1:1.
5. the method of claim 2, wherein the chromatographic column model in step (2) is COSMOSIL5C18-MS-II, 20mm x 250mm.
6. Use of the croconic acid cyanine dye of claim 1 for the preparation of a tumor imaging reagent.
7. The use of claim 6, wherein the imaging agent is a photoacoustic imaging agent or a fluorescent imaging agent.
8. Use of a croconic acid cyanine dye according to claim 1 for the preparation of an inhibitor of tumor cell activity, characterized in that the tumor cell is a liver cancer cell.
9. The use according to claim 8, wherein the tumor cells comprise hepatoma cells HepG-2 and hepatoma cells 7404.
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