Novel photoacoustic-enhancing supramolecular cyanine dye contrast agent, and preparation method and application thereof

Abstract

The invention discloses a novel supramolecular cyanine dye contrast agent for enhancing photoacoustic, and a preparation method and application thereof. Wherein the molecular pair cyanine dye is composed of a cationic indocyanine dye and an anionic tricyanofurancyanine dye. The dye is subjected to J-aggregation in water, so that the absorption wavelength is subjected to red shift to a near-infrared region, and excellent photothermal conversion efficiency (10-80%) and singlet oxygen quantum yield (10-50%) are shown. The photoacoustic imaging performance is excellent, and the intensity of a photoacoustic signal generated under the concentration of 25.0 mu M is 7.1 times that of ICG; the photo-thermal treatment effect is obvious, the photo-thermal conversion efficiency is as high as 66%, and the temperature is rapidly increased to 48 DEG C. In addition, the dye shows excellent light stability and photobleaching resistance, the absorbance is only attenuated by 25% under strong laser, and the dye has good biocompatibility and safety. The multi-mode application comprises photoacoustic and fluorescence imaging, photothermal and photodynamic therapy and the like, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological imaging agents and tumor treatment photosensitizer agents, and relates to a novel photoacoustic enhanced supramolecular cyanine dye contrast agent, a preparation method and application thereof. Background Art

[0002] Photoacoustic imaging (PAI) has brought new opportunities for the development of therapeutic diagnostic imaging technology. In PAI, laser pulses excite light absorbers, and the absorbed laser energy is converted into heat by endogenous or exogenous contrast agents in the tissue. This leads to transient thermoelastic expansion and the generation of corresponding acoustic waves. These acoustic waves can be detected and analyzed by ultrasound transducers to ultimately produce images. PAI is a novel biomedical imaging technique that can provide in vivo imaging information with significant tissue penetration while maintaining high image contrast and spatial resolution. Compared with fluorescence imaging, PAI has excellent tissue penetration (up to 5-6 cm), making it particularly beneficial for deeper tissue imaging with significant clinical applications. On the other hand, photothermal therapy (PTT) is also a rapidly emerging cancer treatment method based on photothermal agent-induced local heating under light irradiation. Compared with photodynamic therapy, which relies on the combined action of photosensitizers, oxygen, and light, PTT is more useful for the ablation of solid tumors with hypoxic microenvironments due to its oxygen-independent nature.

[0003] However, the prior art has the following defects:

[0004] Weak photoacoustic signals: Existing near-infrared dyes such as IR-800-CW, indocyanine green (ICG), and methylene blue (MB), although widely used in fluorescence imaging, perform poorly in generating photoacoustic signals. In particular, ICG has a weak photoacoustic signal and is susceptible to photobleaching and thermal decomposition.

[0005] Lack of optimized design guidelines: There is currently limited understanding of how to design efficient photoacoustic agents, and most studies rely on knowledge of fluorescence, which leads to an inverse relationship between fluorescence quantum yield and photoacoustic activity and cannot effectively guide the design of photoacoustic agents.

[0006] Limited clinical application: Due to the deficiencies of existing dyes in photostability, photoacoustic signal intensity and photothermal effect, their practical application in deep tissue imaging and photothermal therapy is limited.

[0007] Lack of versatility: Existing dyes are difficult to simultaneously meet multiple requirements such as high photoacoustic signal, strong photothermal effect and good biocompatibility, thus limiting their application potential in complex medical environments.

[0008] Photoacoustic imaging relies heavily on commercially available near-infrared dyes, including IR-800-CW, indocyanine green (ICG), or methylene blue (MB). However, these dyes have significant drawbacks:

[0009] Not all fluorescent NIR dyes present measurable photoacoustic signals. For example, although Rhodamine 800 has strong fluorescence, it cannot generate effective photoacoustic signals. Although photoacoustic imaging or photothermal agents can rely on traditional NIR fluorescent dyes, the basic understanding of the structural features that generate photoacoustic signals is limited. In general, the relationship between the generation of photoacoustic signals and structural features is based on fluorescence knowledge, so there is a lack of guiding principles for optimizing the design of photoacoustic agents. For example, compounds with high benzene ring density, electron-donating groups, and central ring rigidity tend to show fluorescent properties. However, the generation of fluorescence and photoacoustic signals are different pathways of energy dissipation after absorbing light, so the fluorescence quantum yield and photoacoustic activity are inversely proportional. Since fluorescence is caused by radiative decay, while photoacoustic activity is caused by non-radiative decay, such as heat generation, there may be components that cause increased photoacoustic activity at the expense of fluorescence. In contrast, Rhodamine 800 generates a large amount of fluorescence signal but cannot generate a measurable photoacoustic signal. Third, satisfactory photostability of PAI agents is essential for long-term high-quality imaging. Indocyanine green (ICG) is an FDA-approved organic dye that has been widely used in PAI research. Unfortunately, ICG itself produces weak photoacoustic signals and is susceptible to photobleaching and thermal decomposition, resulting in a significant reduction in the PA signal during imaging over a long period of time. These limitations restrict the application of existing dyes in deep tissue imaging and photothermal therapy.

[0010] Therefore, it is crucial to develop organic NIR photothermal agents (PTAs) with excellent photostability, efficient photoacoustic imaging capability, enhanced photothermal effect, and outstanding biocompatibility. Summary of the invention

[0011] The present invention aims at the deficiencies of the above-mentioned prior art; the present invention aims to solve the limitations of existing dyes in terms of photoacoustic signal intensity, photostability and photothermal effect by introducing specific tricyanofuranyl groups, indole groups and carefully designed molecular structures, and provides a type of contrast agent containing cationic indocyanine dye and anionic tricyanofuranyl cyanine dye molecular pairs, specifically molecular pair cyanine dyes, and discloses its preparation method and application. This type of dye molecular pair exhibits excellent performance in photoacoustic imaging and optical therapy.

[0012] The present invention successfully solves the limitations of the prior art by introducing specific substituents on the indole group and carefully designed molecular structures: the present invention uses anionic tricyanofuranocyanine dyes as main ligands and different cationic indole cyanine dyes through electrostatic interactions to synthesize a class of molecular paracyanine dyes. The introduction of different substituents on the nitrogen atom of the cationic cyanine dye indole can not only change the aggregation state of the molecular paracyanine dye, but also change the absorption wavelength and fluorescence properties of the molecular paracyanine dye, and enhance the molecular photoacoustic signal and photothermal generation ability, and optimize the optical properties. The research results show that the molecular paracyanine dye has excellent photoacoustic imaging effect and high photothermal conversion efficiency, and is a photoacoustic imaging contrast agent that can be used for photothermal therapy.

[0013] The technical solution of this application is as follows:

[0014] The first aspect of the present application is to protect a class of cationic and anionic molecular paracyanine dyes; molecular paracyanine dyes composed of cationic indocyanine dyes and anionic tricyanofuranocyanine dyes, wherein the dyes have a structure of general formula I:

[0015]

[0016] In the general formula I,

[0017] R 1 and R 2 Each independently selected from hydrogen, an alkyl group having 1-18 carbon atoms, a carboxyalkyl group having 1-18 carbon atoms, an alkylhydroxyl group having 1-18 carbon atoms, or a haloalkyl group having 1-18 carbon atoms, an aryl group, an arylcarboxylic acid group, an alkyl sulfonate group, an aryl sulfonate group, an alkyl sulfonate salt, or an aryl sulfonate salt; further selected from a carboxyalkyl group having 1-6 carbon atoms; more preferably selected from an aryl group having 1-6 carbon atoms, a linear alkyl group having 1-6 carbon atoms (such as one of methyl, ethyl, propyl, benzyl, and ethylbenzene);

[0018] n1 is 1 or 2, n2 is 1 or 2;

[0019] In the technical solution described above, it is further preferred that the anionic tricyanofuranocyanine dye is selected from one of the following structural formulas:

[0020]

[0021] X + Selected from H, Na + , K + , Ca 2+ One of;

[0022] In the technical solution described above, it is further preferred that the cationic indocyanine dye is selected from one of the following structural formulas:

[0023]

[0024] Y - Selected from halogen ions, ClO 4 - , BF 4 - , CH 3 COO - CF 3 COO - or OTs - .

[0025] For the technical solution described above, it is further preferred that the molar ratio of the molecular pair cyanine dye, the cationic indocyanine dye and the anionic tricyanofuran cyanine dye is 1:0.1-10:1. The molar ratio of the cationic indocyanine dye and the anionic tricyanofuran cyanine dye is preferably 1:1-5:1, and most preferably 1:2-2:1. This ratio ensures an effective interaction between the two dyes, thereby optimizing their photophysical properties.

[0026] In the technical solution described above, it is further preferred that the singlet oxygen quantum yield of the dye is 10-50%. The preferred range is 20-40%. The most preferred range is 30-40%.

[0027] In the technical solution described above, it is further preferred that the light-to-heat conversion efficiency of the dye is 10-80%. The preferred range is 30-70%. The most preferred range is 40-70%.

[0028] The second aspect of the present application is to protect the preparation method of the molecular paracyanine dye described above, comprising the following steps:

[0029] (1) The molar ratio of the cationic indocyanine dye to the anionic tricyanofuranocyanine dye in the molecular pair cyanine dye is 1:0.1-10:1.

[0030] (2) The mixing of the cationic indocyanine dye and the anionic tricyanofuranocyanine dye in the molecular paracyanine dye is carried out in water or an organic solvent. The organic solvent includes but is not limited to any one or more of acetonitrile, methanol, ethanol, dichloromethane, chloroform, DMSO, and DMF. Preferably, water is used as the solvent.

[0031] The molecular paracyanine dyes described in the present invention have broad application prospects, especially in the fields of biological photoacoustic imaging and preparation of optical therapeutic agents. Specifically, they are suitable for the following application scenarios:

[0032] Cell imaging: The dye can be used for high-contrast fluorescence imaging or photoacoustic imaging of the internal structure of cells, providing clear cell images.

[0033] Protein labeling: As an efficient labeling tool, this dye can be used to specifically label proteins or other biomolecules for precise tracking and analysis.

[0034] Nucleic Acid Labeling: These dyes are also suitable for labeling DNA or RNA for gene expression studies or diagnostic testing.

[0035] Photothermal therapy (PTT) or photodynamic therapy (PDT): The dye can be used as a highly efficient contrast agent to selectively kill tumor cells or inhibit cancer cell growth under near-infrared light irradiation, using the generated heat or reactive oxygen species.

[0036] The molecular paracyanine dye of the present invention can cover the range of 600-1200nm for fluorescence or photoacoustic imaging emission wavelength in detection applications, and can be selected in the range of 660-1200nm for excitation wavelength in therapeutic applications. Preferably, the emission wavelength and the excitation wavelength are respectively between 600 and 1000nm, and the performance within this range is particularly outstanding. More preferably, when the emission wavelength and the excitation wavelength are between 600 and 900nm, the dye exhibits the highest sensitivity and the most effective therapeutic effect.

[0037] The present invention has the following beneficial effects:

[0038] The anionic cationic molecular paracyanine dye of the present invention has near-infrared light absorption, strong photothermal conversion ability and strong photoacoustic response. It has strong applicability, can realize various types of combinations and performance improvement, and has flexible and diverse preparation methods.

[0039] Excellent photoacoustic imaging performance

[0040] The photoacoustic signal intensity generated by the dye of the present invention at a concentration of 25.0 μM is 7.1 times higher than that of ICG, while the photoacoustic signal of the anionic tricyanofuranocyanine dye alone is only 0.8 times that of ICG. In the near-infrared region (700-800nm), the photoacoustic signal is highly efficient and can achieve deep and clear imaging.

[0041] Enhanced photothermal therapy effects

[0042] The photothermal conversion efficiency is as high as 66%, far exceeding ICG (20%) and the single anionic tricyanofuranocyanine dye (38%). 2 Under laser power, the temperature rises rapidly to 48°C, effectively killing tumor cells. It is suitable for the treatment of solid tumors in hypoxic microenvironments.

[0043] Optimized photodynamic therapy performance

[0044] The high singlet oxygen quantum yield can reach 37%, which is significantly higher than ICG (7.7%), enhancing the killing effect on cancer cells.

[0045] Excellent photostability

[0046] Resistance to photobleaching and thermal decomposition: Under strong laser power (0.6W / cm 2 ), after 4 minutes of continuous irradiation, the absorbance of the cyanine dye decayed by 25%, while that of ICG decayed by more than 72%.

[0047] Good biocompatibility and safety

[0048] Low toxicity: At a concentration of 10 μM, the toxicity to normal cells is extremely low, and the cell survival rate remains above 85%.

[0049] Multimodal imaging and therapy

[0050] It is suitable for photoacoustic imaging, fluorescence imaging, photothermal therapy and photodynamic therapy, providing comprehensive diagnosis and treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The absorption and emission spectra of the molecular pair 1, compound 1.2 and compound 1.4 in methanol (MeOH), ethanol (EtOH) and water (c = 2.0 μM); it shows that the molecular pair undergoes J-aggregation in water, which can red-shift its absorption wavelength to the near infrared 790 nm, which is 52 nm red-shifted compared to the monomer anionic cyanine dye.

[0052] Figure 2 This is the absorption spectrum of the molecular pair 2-5 and its corresponding single-molecule dye in water, which indicates that the aggregation of the molecular pair is related to the lipid solubility of the cationic indocyanine dye.

[0053] Figure 3 The molecular pair 1, compound 1.2, ICG and compound 1.4 were subjected to a power density of 0.1 W cm in water. -2 The absorption changes under 730nm laser irradiation indicate that the molecular stability of cyanine dyes is better than that of monomeric cyanine dyes and commercial ICG.

[0054] Figure 4 DCFH, compound 1.2, ICG and molecular pair 1 were reacted in water at a power density of 0.1 W cm -2 The fluorescence change of DCFH under 808nm laser irradiation shows that the molecular ability to generate reactive oxygen species for cyanine dyes is superior to that of monomeric cyanine dyes and commercial ICG.

[0055] Figure 5 (A) Under 808nm laser irradiation (0.6W cm -2 (A) Photothermal conversion rate of molecular pair 1 at different concentrations (10.0-40.0 μM) under different exposure intensities (0.1-0.6 W cm-2 ) under 808 nm laser irradiation. (C) Molecular pairs 1, compound 1.2 and compound 1.4 (20.0 μM) and water under 808 nm laser irradiation (0.6 W cm -2 ). (D) Photothermal stability study of the molecular pair 1 (20.0 μM) during three heating-cooling processes. (E) Photothermal stability of the molecular pair 1 (20.0 μM) under 808 nm laser irradiation (0.6 W cm -2 (E) The heat transfer time constant (τs) determined from the time-temperature data of the cooling period, and the calculated results of the photothermal conversion efficiency (η) of the molecular pair 1. This shows that the photothermal conversion efficiency of the molecular pair cyanine dye is much better than that of the commercial ICG and the single cyanine dye.

[0056] Figure 6 In vitro PA images of molecular pair 1, compound 1.2, and ICG in water at different concentrations (0–100 μM). The image of molecular pair 1 was collected under 780 nm light excitation; the image of compound 1.2 was collected under 760 nm light excitation; the image of ICG was collected under 772 nm light excitation. (H) PA signal generation efficiency (SGE) of molecular pair 1, compound 1.2, and ICG at constant molar concentration, showing relative dye “loudness” (ICG set to 1); this shows that molecular pair cyanine dyes can greatly enhance the photoacoustic signal of single-molecule cyanine dyes and can be used for photoacoustic imaging.

[0057] Figure 7 Figure 3 is the cell survival rate of 4T1 cells incubated with different concentrations of compound 1.2 or molecule pair 1 with or without laser irradiation, indicating that molecule pair 1 has excellent PTT efficacy.

[0058] Figure 8 The PA and ultrasound superimposed images of compound 1.2 or molecular pair 1 at the tumor site show the change of PA signal over time. The PA signal was collected at 820nm (for molecular pair 1) and 760nm (for compound 1.2) light excitation. Note: Only the tumor area of ​​interest is shown in the PA image. Gray and color bars represent ultrasound and PA intensity, respectively. This shows that the imaging effect of molecular pair 1 in the tumor environment is far better than that of single molecule cyanine dye in the tumor. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below. Unless otherwise specified, the terms used herein have the following meanings.

[0060] The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.

[0061] The term "alkyl group" used in the present invention includes straight-chain alkyl groups and branched-chain alkyl groups.

[0062] Y- is used herein to represent a negative ion, which can be any suitable negative ion, including inorganic negative ions and organic negative ions, and can be exemplified by but not limited to halogen ions, ClO 4- PF 6- , BF 4- , CH 3 COO - CF 3 COO - or OTs - .

[0063] The salt forms of the dyes described herein can be used directly for staining biological samples. Alternatively, in one embodiment, the compounds of the present invention can be used as derivatives of the compounds of formula I.

[0064] The instruments and equipment used in the examples are:

[0065] In the column chromatography process of the present invention, column chromatography silica gel with 200-300 mesh and 100-200 mesh purchased from Qingdao Meigao Group Co., Ltd. and analytical pure quartz sand with 20-40 mesh purchased from Tianda Chemical Reagent Factory are used. Low temperature reaction is achieved by using a magnetic stirring low temperature constant temperature reactor. The reverse phase purification chromatograph is CHEETAH rapid purification preparation chromatograph produced by Bona Ajel Technology Co., Ltd.

[0066] The dye absorption and emission spectra were measured using Agilent's Cary 60 UV-visible spectrophotometer and Cary Eclipse fluorescence spectrophotometer. The dye's absolute fluorescence quantum yield was measured using a C11347 absolute fluorescence quantum yield instrument from Hamamatsu Photonics Trading (China) Co., Ltd.

[0067] Example 1 Preparation of Molecular Pair 1

[0068]

[0069] Example 1.1

[0070]

[0071] 3-Hydroxy-3-methylbutan-2-one (3mmol, 0.306g), malononitrile (6mmol, 0.396g) and sodium ethoxide (0.45mmol) were mixed in ethanol (30mL) and refluxed at 80℃ for 3 hours. The mixture was poured into distilled water (200mL); extracted with dichloromethane (50mL×3), and the organic phase was separated and collected. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. Finally, CH 2 CI 2The crude product was separated by silica gel column chromatography using 1% / MeOH (v / v, 100:1). The obtained solid was crystallized in ethanol (528 mg, 88%) to give yellow solid compound 1.1.

[0072] Example 1.2

[0073]

[0074] Compound 1.1 of Example 1.1 (4.4 mmol, 2.2 eq), malondialdehyde diphenylamine hydrochloride (0.52 g, 2.0 mmol, 1.0 eq) and anhydrous NaOAc (0.37 g, 4.4 mmol, 2.2 eq) were mixed with 20 mL of acetic anhydride and heated to 110° C. for 2 h under nitrogen. The reaction solution was then cooled and poured into 100 mL of saturated brine, followed by extraction with DCM (3×50 mL) and then added to anhydrous Na 2 Dry over SO4 and evaporate in vacuo. The residue was purified by chromatography (silica gel) using 80-40:1 DCM / methanol (v / v) as the eluting solvent. After removal of the solvent, a solid product, compound 1.2(A1) (0.47 g, yield 52%) was obtained.

[0075] Example 1.3

[0076]

[0077] 2,3,3-Trimethyl-3H-indoline (7.955 g, 50 mmol) and iodoethane (11.690 g, 75 mmol) were added to a 100 mL single-mouth round-bottom flask containing 25 mL of toluene, heated to reflux under nitrogen protection, and the reaction was stopped after 12 hours of continuous reaction. After cooling to room temperature, 50 mL of ether was added to the reaction solution, filtered, and the filter cake was washed with ether. Natural air drying was performed to obtain 11.173 g of a crude product of pink solid powder compound 1.3 (1-ethyl-2,3,3-trimethyl-3H-indoline iodide salt), with a crude yield of 71%.

[0078] Example 1.4

[0079]

[0080] Take compound 1.3 (1-ethyl-2,3,3-trimethyl-3H-indoline iodide) (1.260 g, 4 mmol) of Example 1.3 and malondialdehyde diphenylamine hydrochloride condensation agent (0.516 g, 2 mmol), add them into a 100 mL single-necked flask, then add anhydrous sodium acetate (0.340 g, 4 mmol) and 20 mL of acetic anhydride, heat to 110°C under a nitrogen atmosphere, the color of the solution quickly turns blue, stop heating after half an hour of reaction, and wait until the temperature reaches room temperature. The reaction mixture was washed with saturated brine, and the organic layer was separated. The aqueous layer was extracted with dichloromethane for several times, and then combined with the previous organic layer, dried over anhydrous sodium sulfate, filtered, and the solvent was rotary evaporated to dryness. The crude product was separated and purified by silica gel chromatography and rinsed with anhydrous methanol / dichloromethane (1 / 90, v / v) as a developing agent. The blue component was collected and evaporated to dryness to obtain a blue dye with a metallic luster. 0.753 g of product compound 1.4 was obtained with a yield of 70%.

[0081] Making molecular pair 1

[0082]

[0083] Method 1

[0084] Weigh 2.70 mg of compound 1.4 and 2.28 mg of compound 1.2, dissolve in 1.0 mL of DMSO, mix well, filter using a 0.45 μm organic filter membrane to obtain a 5.0 mM molecular pair 1 mother solution in a brown sample bottle, and store in a refrigerator (0-4° C.) for later use.

[0085] Method 2

[0086] Weigh 5.38 mg of compound 1.4, dissolve it in 1.0 mL of DMSO to obtain a mother solution A1 with a concentration of 10.0 mM; weigh 4.56 mg of compound 1.2, dissolve it in 1.0 mL of DMSO to obtain a mother solution B1 with a concentration of 10.0 mM, then take 0.5 mL of mother solution A1 and 0.5 mL of mother solution B1 and mix them thoroughly, and filter them using a 0.45 μm organic filter membrane to obtain a molecular pair 1 mother solution with a concentration of 5.0 mM.

[0087] Example 1.5

[0088]

[0089] Compound 1.1 of Example 1.1 (4.4 mmol, 2.2 eq), pentadienaldehyde diphenylamine hydrochloride (0.57 g, 2.0 mmol, 1.0 eq) and anhydrous NaOAc (0.37 g, 4.4 mmol, 2.2 eq) were mixed with 20 mL of acetic anhydride and heated to 110° C. for 2 h under nitrogen. The reaction solution was then cooled and poured into 100 mL of saturated brine, followed by extraction with DCM (3×50 mL) and then added to anhydrous Na 2 Dry over SO4 and evaporate in vacuo. The residue was purified by chromatography (silica gel) using 80-40:1 DCM / methanol (v / v) as eluting solvent. After removal of the solvent, compound 1.5 (0.40 g, yield 41%) was obtained.

[0090] Example 1.6

[0091]

[0092] Add 2,3,3-trimethyl-3H-indoline (0.79 g, 5 mmol) and iodoethanol (1.29 g, 7.5 mmol) to a 100 mL single-mouth round-bottom flask containing 10 mL of acetonitrile, heat to reflux under nitrogen protection, and continue the reaction for 12 hours before stopping the reaction. Cool to room temperature, add 50 mL of ether to the reaction solution, filter, and wash the filter cake with ether. Natural air drying, a pink solid powder crude product of 1.02 g is obtained, and the crude yield is 62%.

[0093] Example 1.7

[0094]

[0095] Take compound 1.6 (1.26 g, 2 mmol) of Example 1.6 and malondialdehyde diphenylamine hydrochloride condensation agent (0.26 g, 1 mmol), add to a 100 mL single-necked flask, then add anhydrous sodium acetate (0.17 g, 2 mmol) and 10 mL acetic anhydride, heat to 110 ° C under nitrogen atmosphere, the solution color quickly turns blue, stop heating after half an hour of reaction, and wait until the temperature reaches room temperature. Wash with saturated brine, separate the organic layer, extract the water layer with dichloromethane several times, then combine with the previous organic layer, dry with anhydrous sodium sulfate, filter, and evaporate the solvent by rotary evaporation. The crude product is separated and purified by silica gel chromatography and rinsed with anhydrous methanol / dichloromethane (1 / 90, v / v) developing agent, collect the blue component, and evaporate to dryness to obtain a blue dye with metallic luster, and obtain 0.36 g of product with a yield of 64%.

[0096] The following compounds were prepared in the same manner as in Examples 1.6 and 1.7. Specific examples of the compounds represented by the general formula B1 are given below, but the present invention is not limited to these specific examples.

[0097]

[0098] Example 1.8

[0099]

[0100] Take compound 1.3 (1-ethyl-2,3,3-trimethyl-3H-indoline iodide) (1.260 g, 4 mmol) of Example 1.3 and pentadienaldehyde diphenylamine hydrochloride (0.57 g, 2.0 mmol, 1.0 eq), add them into a 100 mL single-necked flask, then add anhydrous sodium acetate (0.340 g, 4 mmol) and 20 mL of acetic anhydride, heat to 110°C under a nitrogen atmosphere, the color of the solution quickly turns blue, stop heating after half an hour of reaction, and wait until the temperature reaches room temperature. The reaction mixture was washed with saturated brine, and the organic layer was separated. The aqueous layer was extracted with dichloromethane for several times, and then combined with the previous organic layer, dried over anhydrous sodium sulfate, filtered, and the solvent was rotary evaporated to dryness. The crude product was separated and purified by silica gel chromatography and rinsed with anhydrous methanol / dichloromethane (1 / 80, v / v) as a developing agent. The blue component was collected and evaporated to dryness to obtain a brown dye with a metallic luster. 0.27 g of product was obtained with a yield of 48%.

[0101] The following compounds were prepared in the same manner as in Examples 1.6 and 1.8. Specific examples of the compounds represented by the general formula B2 are given below, but the present invention is not limited to these specific examples.

[0102]

[0103] Example 1.9

[0104] 1,1,2-Trimethyl-1H-benzo[e]indole (1.05 g, 5 mmol) and iodoethane (1.56 g, 10 mmol) were added to a 100 mL single-mouth round-bottom flask containing 25 mL of toluene, heated to reflux under nitrogen protection, and the reaction was stopped after 12 hours of continuous reaction. After cooling to room temperature, 50 mL of ether was added to the reaction solution, filtered, and the filter cake was washed with ether. Natural air drying was performed to obtain 1.39 g of a pink solid powder compound 1.9 crude product, with a crude yield of 76%.

[0105] Example 1.10

[0106]

[0107] Take the compound 1.9 of Example 1.9 (0.36 g, 1 mmol) and malondialdehyde diphenylamine hydrochloride condensation agent (0.13 g, 0.5 mmol), add them to a 100 mL single-necked flask, then add anhydrous sodium acetate (0.080 g, 1 mmol) and 5 mL of acetic anhydride, heat to 110 ° C under a nitrogen atmosphere, the solution color quickly turns blue, stop heating after half an hour of reaction, and wait until the temperature reaches room temperature. Wash with saturated brine, separate the organic layer, extract the aqueous layer with dichloromethane several times, then combine with the previous organic layer, dry with anhydrous sodium sulfate, filter, and evaporate the solvent to dryness by rotary evaporation. The crude product is separated and purified by silica gel chromatography and rinsed with anhydrous methanol / dichloromethane (1 / 90, v / v) developing agent, collect the blue component, and evaporate to dryness to obtain a blue dye with a metallic luster, to obtain 0.16 g of product, with a yield of 50%.

[0108] The following compounds were prepared in the same manner as in Examples 1.9 and 1.10. Specific examples of the compounds represented by the general formula B3 are given below, but the present invention is not limited to these specific examples.

[0109]

[0110] Example 1.11

[0111]

[0112] Take the compound 1.9 of Example 1.9 (0.36 g, 1 mmol) and glutaraldehyde diphenylamine hydrochloride condensation agent (0.14 g, 0.5 mmol), add them to a 100 mL single-necked flask, then add anhydrous sodium acetate (0.080 g, 1 mmol) and 5 mL of acetic anhydride, heat to 110 ° C under a nitrogen atmosphere, the solution color quickly turns blue, stop heating after half an hour of reaction, and wait until the temperature reaches room temperature. Wash with saturated brine, separate the organic layer, extract the aqueous layer with dichloromethane several times, then combine with the previous organic layer, dry with anhydrous sodium sulfate, filter, and evaporate the solvent to dryness by rotary evaporation. The crude product is separated and purified by silica gel chromatography and rinsed with anhydrous methanol / dichloromethane (1 / 90, v / v) developing agent, collect the blue component, and evaporate to dryness to obtain a blue dye with a metallic luster, to obtain 0.156 g of the product, with a yield of 47%.

[0113] The following compounds were prepared in the same manner as in Examples 1.9 and 1.11. Specific examples of the compounds represented by the general formula B4 are given below, but the present invention is not limited to these specific examples.

[0114]

[0115] Example 1.12

[0116]

[0117] Take compound 1.3 (1-ethyl-2,3,3-trimethyl-3H-indoline iodide) (1.260 g, 4 mmol) of Example 1.3 and N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride (0.71 g, 2.0 mmol, 1.0 eq), add them to a 100 mL single-necked flask, then add anhydrous sodium acetate (0.340 g, 4 mmol) and 20 mL of acetic anhydride, heat to 110°C under a nitrogen atmosphere, the color of the solution quickly turns blue, stop heating after half an hour of reaction, and wait until the temperature reaches room temperature. The reaction mixture was washed with saturated brine, and the organic layer was separated. The aqueous layer was extracted with dichloromethane several times and then combined with the previous organic layer, dried over anhydrous sodium sulfate, filtered, and the solvent was rotary evaporated to dryness. The crude product was separated and purified by silica gel chromatography and rinsed with anhydrous methanol / dichloromethane (1 / 80, v / v) as a developing agent. The blue component was collected and evaporated to dryness to obtain a brown dye with a metallic luster. 0.47 g of product was obtained with a yield of 37%.

[0118] The following compounds were prepared in the same manner as in Examples 1.9 and 1.12. Specific examples of the compounds represented by the general formula B5 are given below, but the present invention is not limited to these specific examples.

[0119]

[0120] Example 1.13

[0121]

[0122] Compound 1.9 (0.36 g, 1 mmol) of Example 1.9 and N-[(3-(anilinomethylene)-2-chloro-1-cyclohexene-1-yl)methylene]aniline hydrochloride (0.18 g, 0.5 mmol, 1.0 eq) were added to a 100 mL single-necked flask, followed by anhydrous sodium acetate (0.080 g, 1 mmol) and 5 mL of acetic anhydride. The mixture was heated to 110 °C under a nitrogen atmosphere, and the color of the solution quickly turned blue. After half an hour of reaction, stop heating and wait until the temperature reaches room temperature. Wash with saturated brine, separate the organic layer, extract the water layer with dichloromethane several times, then combine with the previous organic layer, dry with anhydrous sodium sulfate, filter, evaporate the solvent by rotary evaporation, separate and purify the crude product by silica gel chromatography, rinse with anhydrous methanol / dichloromethane (1 / 80, v / v) developing agent, collect the blue component, evaporate to dryness to obtain a brown dye with metallic luster, and obtain 0.30g of product with a yield of 41%.

[0123] The following compounds were prepared in the same manner as in Examples 1.9 and 1.13. Specific examples of the compounds represented by the general formula B6 are given below, but the present invention is not limited to these specific examples.

[0124]

[0125] Example 2 Preparation of Molecular Pair 2

[0126] Molecular pair 2 was prepared according to method 1 in Example 1.

[0127]

[0128] Weigh 2.85 mg of compound 1.7 and 2.28 mg of compound 1.2, dissolve in 1.0 mL of DMSO, mix well, filter using a 0.45 μm organic filter membrane to obtain a 5.0 mM molecular pair 2 mother liquor in a brown sample bottle, and store in a refrigerator (0-4° C.) for later use.

[0129] The following molecular pair compound contrast agents were prepared in the same manner as in Example 2. Specific examples of the compounds represented by the general formula I are given below, but the present invention is not limited to these specific examples.

[0130] Molecular pair 3

[0131]

[0132] Molecular pair 4

[0133]

[0134] Molecular pair 5

[0135]

[0136] Molecular pair 6

[0137]

[0138] Molecular pair 7

[0139]

[0140] Molecular pair 8

[0141]

[0142] Example 3

[0143] UV-visible absorption and fluorescence spectra of compounds and molecules for contrast agents

[0144] When testing the UV-visible absorption spectrum and fluorescence spectrum, use a micropipette to measure 1.2 μL of the probe stock solution and dissolve it in a quartz cuvette containing 3 mL of the solvent to be tested, mix well, and obtain a probe concentration of 2.0 μmol / L for the absorption spectrum and fluorescence emission spectrum test. All tests were completed at 25°C.

[0145] like Figure 1 As shown, compounds 1.2 and 1.4 exhibited individual cyanine dye absorption and emission spectra in both water and ethanol. Compound 1.4 exhibited sharp and strong absorption at 638 nm in water (ε = 22.6 × 10 4 M -1 cm -1 ) and strong fluorescence at 657nm (Φ F =9.7%). Compound 1.2 at 738nm (ε=18.7×10 4 M -1 cm -1 ) shows sharp and strong absorption at 762nm and strong fluorescence at 762nm (Φ F =7.6%) in water. When the two form a molecular pair, they show a single cyanine dye peak in methanol, but aggregate in water. Compound 1.2 shows two significantly broadened absorption peaks: one at 638-644nm for compound 1.4 and the other at 790nm for compound 1.2. Compared with the single molecule spectrum of compound 1.2, the latter shows a significant red shift of 52nm. These data strongly indicate that the compound 1.2 component in molecular pair 1 forms a J-type aggregate in water, which partially shows a wavelength red shift and can achieve near-infrared absorption capacity. Compound 1.4 partially undergoes H aggregation and a wavelength blue shift. In addition, the molecular pair has almost no fluorescence in water, indicating fluorescence quenching, which may enhance photoacoustic (PA) emission and photothermal performance.

[0146] Example 4

[0147] UV-visible absorption spectra of compounds and molecules for contrast agents 2-5

[0148] When testing the UV-visible absorption spectrum, the test method is consistent with that in Example 3. All tests are completed at 25°C.

[0149] like Figure 2As shown, the cationic indocyanine dye of unimolecular and the unimolecular anionic cyanine dye compound 1.4 all show independent cyanine dye absorption spectrum in water.After both form molecular pairs in water, the absorption peak of both cationic cyanine dye and anionic cyanine dye all declines, indicating that it then gathers in water.The absorption peak of molecule pairs 2,3 and 5 at 800nm ​​long wavelength changes differently, and the intensity order of absorbance value at 800nm ​​is: molecule pairs 2<molecule pairs 5<molecule pairs 3, which may be closely related to the substituent group of cationic cyanine dye.The cationic indocyanine dye substituent group is water-soluble, and the formed molecule pairs absorb weakly at 800nm, and the cationic indocyanine dye substituent group is lipophilic, and the formed molecule pairs absorb strongly at 800nm.In addition, molecule pairs 4 absorb weaker at long wavelength, but form strong broad absorption peak at 600-700nm, indicating that it forms H and gathers.

[0150] Example 5

[0151] Photostability test of compounds 1.2, 1.4, molecular pair 1 and ICG

[0152] Dye photostability experiment: The photostability of the synthesized dye molecules was evaluated by using a photostability test device. 3 mL of water was added to a sealable quartz cuvette, and then 3.0 μL of dye stock solution was measured with a micropipette and added to the cuvette. The seal was closed and mixed evenly. The dye sample was prepared into 5.0 μmol L -1 The solution was prepared and the initial absorbance of the dye sample was measured using a UV-visible spectrophotometer. Then, it was irradiated with 730nm light and the absorbance was measured every 1min. It can be observed that as the illumination time increases, the absorbance of the dye decreases. Finally, the photostability of the dye is compared based on the rate of decrease of the dye absorbance.

[0153] The results are as follows Figure 3 As shown, under 730nm illumination, after 4 minutes, the absorbance of the molecular pair 1 only decreased by 26%, while the absorbance of the monomer anionic cyanine dye decreased by 65%, and the commercial ICG decreased by 42%. This shows that the stability of the molecular pair cyanine dye is greatly improved compared with the monomer and commercial ICG. Under 808nm illumination, after 4 minutes, the absorbance of the molecular pair 1 only decreased by 25%, and the commercial ICG decreased by 79%. This shows that the stability of the molecular pair cyanine dye is also greatly improved compared with the commercial ICG under long-wavelength 808nm illumination. This also further shows that the molecular pair cyanine dye has better photostability and is more conducive to subsequent applications.

[0154] Example 6

[0155] Photostability test of compounds 1.2, 1.4, molecular pair 1 and ICG

[0156] 2`,7`-Dichlorodihydrofluorescein (DCFH) is a fluorescent probe for measuring reactive oxygen species. When oxidized by ROS to 2',7'-dichlorofluorescein hydrogen probe (2',7'-dichlorofluorescein, DCF), it emits strong fluorescence. DCFH has better lipid solubility and membrane permeability, and is used as a probe for detecting intracellular reactive oxygen species (ROS) (Ex / Em=488 / 525nm). Therefore, after illumination, the ability of the photosensitizer to generate ROS can be qualitatively reflected by the degree of enhancement of the green fluorescence of DCF.

[0157] Operation steps: A 1×1 cm cuvette was prepared with an aqueous solution containing 5 μM dye and 5 μM DCFH, and then irradiated with 808 nm laser (optical power: 100 mW / cm 2 ) irradiated the cuvette, and measured the fluorescence spectrum of the DCFH solution every 1 min (excitation wavelength: 510 nm; slits: 5 nm, 10 nm). The presence or absence of ROS was determined by whether the fluorescence intensity of the DCFH aqueous solution increased at 521 nm. The control group was a DCFH aqueous solution without dye.

[0158] The experimental results are as follows Figure 4 As shown, under the same conditions, the control group DCFH and the DCFH aqueous solution containing compound 1.2 only have a weak fluorescence increase at 521nm, while the DCFH aqueous solution containing ICG has a certain fluorescence increase at 521nm, indicating that the ROS generation ability of ICG is better than that of the single molecule compound 1.2; the DCFH aqueous solution containing the molecular pair 1 has a significant fluorescence increase at 521nm, and its growth rate is 4.8 times that of ICG, indicating that the ability of the molecular pair 1 to generate ROS under light conditions is significantly improved compared with the single molecule compound 1.2 and compound 1.4 and commercial ICG. It shows that the patented molecular pair can promote the active oxygen generation ability of the molecule.

[0159] Example 7

[0160] Photothermal performance test of compound 1.2, molecular pair 1 and ICG

[0161] The infrared thermal imager was used to measure the -2 , 10 min treatment of the compound, molecular pair 1 and ICG aqueous solution temperature changes. The sample temperature was recorded every 30 seconds using laser irradiation. Aqueous solutions of molecular pair 1 with different concentrations (0, 10.0, 20.0, 40.0 μM) were prepared and irradiated with 808 nm laser (0.6 W cm -2 ) and record the temperature every 30 seconds.

[0162] The experimental results are as follows Figure 5 As shown, under the same conditions, the aqueous solution of the molecular pair 1 can be heated to 51°C, while ICG can only be heated to 38.9°C, and the single molecule anionic cyanine dye 1.2 can only be heated to 36.5°C, indicating that the photothermal conversion ability of the molecular pair 1 is significantly improved compared with the single molecule compound 1.2 and compound 1.4 and the commercial ICG. This shows that the molecular pair of this patent can promote the photothermal conversion ability of the molecule.

[0163] Example 8

[0164] PA imaging detection

[0165] In vitro PA imaging was performed using a Visualsonics LAZR Vevo system (Fuiifilm, Japan) equipped with a 256-element transducer array (MX550D, 25 to 55 MHz) and a nanosecond pulsed laser (pulse width of 10 ns and repetition rate of 20 Hz). Solutions of compound 1.2 and molecular pair 1 and ICG were enclosed in a transparent polyurethane tube and immersed in a water tank for photoacoustic coupling.

[0166] The experimental results are as follows Figure 6 As shown, contrast agent molecule pair 1 exhibited the strongest PA signal in the concentration range of 0.0 to 100.0 μM. In addition, an excellent linear correlation was observed between the concentration of molecule pair 1 and the PA intensity. The photoacoustic signals of compound 1.2 and commercial ICG were very weak. In order to roughly quantify the PA signal generation efficacy (SGE) using the Thorn threshold method, we selected an intermediate concentration of 25.0 μM, which was linear for all species (ICG was set to 1). It is worth noting that the PA intensity of contrast agent molecule pair 1 is more than 7.1 times higher than that of ICG (higher than single molecule compound 1.2), indicating that it can be used for clinical photoacoustic imaging (PAI) studies. Therefore, the excellent PA response behavior indicates that contrast agent molecule pair 1 has the potential to be used to guide in vivo photothermal therapy (PTT).

[0167] Example 9

[0168] The dark toxicity and phototoxicity of compound 1.2 and molecular pair 1 contrast agents to tumor cells were evaluated by CCK8 assay.

[0169] Specific operation steps: 4T1 cells were digested with trypsin and diluted to a cell suspension with a concentration of 5×103 cells / mL. 100 μL of cell suspension was added to each well of a 96-well plate. After culturing to a density of about 70% at 37°C and 5% CO2, 100 μL of culture medium containing different concentrations of dye was added and placed in an incubator for 1 hour. The dark toxicity control group was incubated for 12 hours without light, and the experimental group was illuminated with 808 nm laser (600 mW cm -2) for 5 min, and then cultured at 37°C for 12 h. Then, the cells were subjected to CCK8 programmed operation and shaken by an ELISA reader for 3 min. The absorbance at 450 nm was measured, and the cell survival rate was calculated according to the following formula.

[0170] Cell viability = ([OD] 染料组 -〖OD〗 空白组 ) / (〖OD〗 对照组 -〖OD〗 空白组 )×100%

[0171] The experimental results are as follows Figure 7 As shown, the CCK-8 assay was used to evaluate the in vitro phototherapeutic effect of compound 1.2 and molecular pair 1. -2 The survival rate of tumor cells treated with 10.0 μM compound 1.2 was about 80%, while the survival rate of 4T1 cells treated with 10.0 μM molecule pair 1 was only about 20%, highlighting the excellent PTT efficacy of molecule pair 1.

[0172] Example 10

[0173] In vivo photoacoustic signal detection

[0174] In vivo PA imaging was performed using 4T1 tumor-bearing mice, and the mouse model with tumor was anesthetized in oxygen using 2% isoflurane carried through a nose cone, and the body temperature was maintained by a heating stage. Normal saline was injected intraperitoneally as a control, and continuous PA images were collected at intervals after the injection of single molecule compound 1.2 and molecular pair 1 (dose of 200μM, 100μL). The 3D PA mode of the Vevo LAZR system at the corresponding excitation wavelength was used to capture the corresponding signal.

[0175] The experimental results are as follows Figure 8 As shown, the PA imaging effect of single-molecule compound 1.2 in the tumor is very weak, while the molecular pair 1 shows strong PA imaging in the tumor. This shows that the molecular pair 1 has a strong photoacoustic signal in the mouse tumor environment and exhibits excellent photoacoustic imaging enhancement effect.

[0176] The above contents are further elaborations of the present invention in conjunction with the specific preferred embodiments of the present invention, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. Any technician familiar with the profession, without departing from the technical scope of the present invention, makes slight changes or modifications, which will be deemed to be covered by the claims of the present invention.

Claims

1. A type of anionic and cationic molecular cyanine dye, characterized in that: Containing cationic indocyanine dye and anionic tricyanofuranocyanine dye, having the following general formula I structure: In the general formula I, R1 and R2 are each independently selected from hydrogen, an alkyl group having 1-18 carbons, a carboxyalkyl group having 1-18 carbons, an alkylhydroxyl group having 1-18 carbons, or a haloalkyl group having 1-18 carbons, an aryl group, an arylcarboxylate group, an alkyl sulfonate, an aryl sulfonate, an alkyl sulfonate, or an aryl sulfonate; n1 is 1 or 2; n2 is 1 or 2.

2. The molecular paracyanine dye according to claim 1, characterized in that: The anionic tricyanofuranocyanine dye is selected from one of the following structural formulas A1 or A2: X + Selected from H, Na + , K + , Ca 2+ One of them.

3. The molecular paracyanine dye according to claim 1, characterized in that: The cationic indocyanine dye is selected from one of the following structural formulas B1, B2, B3, and B4: Y - Selected from halogen ions, ClO 4- , BF 4- 、CH3COO - CF3COO - or OTs - .

4. The molecular paracyanine dye according to claim 1, characterized in that: The molar ratio of the molecular paracyanine dye, the cationic indocyanine dye and the anionic tricyanofuranocyanine dye is 1:0.1-10:

1.

5. The molecular paracyanine dye according to claim 1, characterized in that: The singlet oxygen quantum yield of the dye is 10-50%.

6. The molecular paracyanine dye according to claim 1, characterized in that: The light-to-heat conversion efficiency of the dye is 10-80%.

7. The method for preparing a molecular paracyanine dye according to claim 1, comprising the following steps: The cationic indocyanine dye and the anionic tricyanofuranocyanine dye are mixed in a molar ratio of 1:0.1-10:

1.

8. The preparation method according to claim 7, characterized in that: The cationic indocyanine dye and the anionic tricyanofuranocyanine dye are mixed in water or an organic solvent; the organic solvent includes but is not limited to any one or more of acetonitrile, methanol, ethanol, dichloromethane, chloroform, DMSO, and DMF.

9. Use of the molecular paracyanine dye as claimed in claim 1 as a contrast agent in the preparation of biological photoacoustic imaging or optical therapy agents.

10. The use according to claim 9, characterized in that: The fluorescence emission wavelength of the molecular cyanine dye during imaging or reagent preparation is 600-1200nm; the excitation wavelength is 660-1200nm.

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