A dicationic cyanine dye, its preparation method and application

Abstract

The application discloses a kind of double cation cyanine dyes and its preparation method and application, the cyanine dye has the structure of general formula I.The double cation cyanine dye described in the application has no heavy atom modification, double cation characteristics, and has near-infrared region one absorption and emission, high specificity mitochondrial targeting ability, high molar extinction coefficient, high efficient photosensitization efficiency ability.Under near-infrared light irradiation, it has excellent tumor cell killing ability, and has good application prospect in antitumor therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fine chemical organic dyes, in particular to a kind of double cation cyanine dye and its preparation method and application. BACKGROUND

[0002] Photodynamic therapy (PDT) is a new type of tumor treatment method, its basic concept is that the energy of photons is absorbed by sensitizers, which induces the sensitizers to jump from ground state to excited state, and the sensitizers in the excited state can produce reactive oxygen in the process of returning to the ground state, so as to kill tumor cells and treat tumors. Because of its unique treatment method, PDT has the advantages of non-invasiveness, spatiotemporal targeting, low toxicity and side effects, and repeated application without drug resistance, which avoids the damage to normal cells and the immune and hematopoietic function of the body caused by traditional cancer treatment methods, and increases the risk of infection at the lesion site. From the mechanism of PDT, light, photosensitizer and molecular oxygen are indispensable, only when the three elements work together can they have the ability to treat tumors.

[0003] As the core and basis of PDT, photosensitizer is the key to promote the clinical application of PDT. As a classic functional dye, cyanine dye has the characteristics of adjustable excitation wavelength, high molar extinction coefficient, good biocompatibility and easy modification of structure, and is an ideal photosensitizer parent. However, cyanine dye also has the disadvantages of low active oxygen quantum yield, poor stability and short triplet state lifetime, which limits its application in tumor treatment. Generally, the conventional design to enhance its intersystem crossing ability and improve the active oxygen yield relies on heavy atom effect, which makes the molecular type of photosensitizer too single. At the same time, heavy atom photosensitizer has the defects of short triplet state lifetime, high cost and other shortcomings. Compared with the above, non-heavy atom photosensitizer can make up for the above defects, and can enrich its types, so it is urgent to explore the design strategy of high-performance non-heavy atom photosensitizer. SUMMARY

[0004] In view of the deficiencies of cyanine dye in photosensitization performance, the present application discloses a kind of double cation cyanine dye, which has great application prospect in the field of fine chemical and life science, especially in the aspect of photosensitizer used for photodynamic therapy. The present application mainly takes conjugation degree, intramolecular electron transfer and double cation characteristics as the regulation mechanism, and designs and develops high-performance double cation cyanine dye with high active oxygen quantum yield, strong photon absorption ability and excellent antitumor effect.

[0005] In order to achieve the above purpose, the technical scheme of the present application is as follows: a kind of double cation cyanine dye, the double cation cyanine dye has the following structure general formula I:

[0006]

[0007] I

[0008] In the general formula I:

[0009] R1 and R2 can be each independently selected from any one of methyl, ethyl, long chain alkyl, benzyl, alkyl sulfonic acid group, tumor targeting group;

[0010] X is selected from any one of dimethyl, oxygen atom, sulfur atom or selenium atom;

[0011] Y is selected from any one of iodine, chlorine, bromine;

[0012] n is selected from any positive integer, preferably, n is selected from any one of 0, 1, 2, 3;

[0013] M1 and M2 can be the same or different, and M1 and M2 are each independently selected from any one of the groups of the following structural formula i or ii:

[0014]

[0015] In the groups of i and ii, R3 can be any one of methyl, ethyl, long chain alkyl, benzyl, alkyl sulfonic acid group, tumor targeting group;

[0016] R4 can be selected from any one of H, Cl, Br, F, carboxylic acid group, sulfonic acid group.

[0017] Further, M1 and M2 are the same and are selected from any one of the groups of the structural formula i or ii.

[0018] Further, the tumor targeting group is selected from any one of rituximab, bevacizumab, female hormone receptor, epidermal growth factor receptor, folic acid, biotin.

[0019] Further, the absorption and emission wavelength of the double cationic cyanine dye in the near infrared I region is 750 ~ 900 nm; the molar extinction coefficient of the double cationic cyanine dye in the near infrared I region is 1.0 ~ 3.0 × 10 5 M -1 cm -1 .

[0020] The preparation method of the double cationic cyanine dye comprises the following steps: using compound II and compound III as raw materials, Knoevenagel condensation reaction occurs in organic acid anhydride, organic base and non-polar solvent, to generate near-infrared double cationic photosensitizer;

[0021]

[0022] Furthermore, the molar ratio of compound II to compound III is 1:1 to 3, the molar ratio of compound II to organic base is 1:4 to 5, and the molar ratio of compound II to organic anhydride is 1:1.5 to 3.

[0023] Furthermore, the organic anhydride is selected from any one of acetic anhydride, propionic anhydride, succinic anhydride, benzoic anhydride, and phthalic anhydride.

[0024] Furthermore, the organic base is selected from triethylamine, pyridine, and N,N-diisopropylethylamine.

[0025] Furthermore, the nonpolar solvent is selected from one of dichloromethane, trichloromethane, and dichloroethane.

[0026] The aforementioned biscationic cyanine dyes are used in bioimaging and medical phototherapy.

[0027] Furthermore, the biscationic cyanine dye is applied to normoxic and hypoxic photodynamic therapy of tumors. The biscationic cyanine dye has highly efficient mitochondrial targeting ability and reactive oxygen species generation under near-infrared light radiation, and can be used for anti-tumor photodynamic therapy.

[0028] In summary, the present invention has the following beneficial effects:

[0029] 1. This application uses benzobispyrrole as a linking intermediate and organically combines the advantages of both dicationic dyes and cyanine dyes to construct a DAD-type dicationic cyanine dye, achieving simultaneous improvement in performance regulation and specific functionalization, which is of great significance for promoting the clinical development of PDT technology;

[0030] 2. The biscationic cyanine dyes prepared in this application are more suitable for long-wavelength absorption and emission scenarios (major absorption spectra located at 780 nm ~ 850 nm, and major emission spectra located at 850 nm ~ 900 nm), and can be well used for deep tissue imaging and tumor treatment; moreover, they have a large molar extinction coefficient in the near-infrared region (1.88 ~ 2.1 × 10⁻⁶). 5 M -1 cm -1 );

[0031] 3. Due to its cationic structure, it has a specific function of targeting mitochondria; it can be excited by near-infrared light and efficiently generate reactive oxygen species (indocyanine green singlet oxygen). 1 It can produce 2 to 3 times more O2 and effectively kill tumor cells.

[0032] Therefore, the dye described in this invention exhibits significant changes in photophysical properties compared to traditional cyanine dyes, and also possesses excellent biocompatibility. In the embodiments of this invention, different concentrations of the photosensitive dye still showed over 90% cell viability after culturing MCF-7 cells for 48 hours, indicating that it does not produce toxic side effects on cells within the working concentration range. Furthermore, under near-infrared light excitation, it can efficiently generate reactive oxygen species for tumor cell killing. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 High-resolution mass spectrometry for 2LBCy5.5;

[0035] Figure 2 High-resolution mass spectra of 2LCy5.5;

[0036] Figure 3 The absorption and emission spectra of Cy5.5, 2LCy5.5, and 2LBCy5.5 photosensitive dyes in dichloromethane are shown.

[0037] Figure 4 For the production of photosensitive dyes 1 O2 destroys DPBF ( 1 The ultraviolet absorption spectra of DPB with 2LBCy5.5 as a photosensitive dye are shown in the figure; (a) is the ultraviolet absorption spectrum of DPB with 2LBCy5.5 as a photosensitive dye; (b) is the ultraviolet absorption spectrum of DPBF with 2LCy5.5 as a photosensitive dye; and (c) is a comparison of the degradation rates of DPBF with the participation of two photosensitive dyes.

[0038] Figure 5 The uptake time of the photosensitive dye in MCF-7 cells and its mitochondrial-targeted fluorescence imaging are shown; (a) shows the uptake time of 2LCy5.5 and its mitochondrial-targeted fluorescence imaging; (b) shows the uptake time of 2LBCy5.5 and its mitochondrial-targeted fluorescence imaging.

[0039] Figure 6 MTT assay of cells with 2LBCy5.5 photosensitive dye. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the appendices in the embodiments of the present invention. Figures 1-6The technical solutions in the embodiments of the present application are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0041] The double-cationic photosensitizing dye (DCPDys) refers to a parent dye having two positive charges, good water solubility, and the positive charge and water solubility characteristics can protect the photosensitizing dye from degradation by various enzymes and enhance the stability in blood circulation. In addition, the double-cationic structure has strong electron-withdrawing ability, which can enhance the separation and transfer ability of intramolecular electrons, is conducive to reducing the energy level between the singlet state and the triplet state, and improves the intersystem crossing efficiency. The DCPDys can also improve the bioavailability by electrostatic interaction and ion complexation channel of the charged biological membrane. Therefore, the development of activatable DCPDys is a powerful way to improve the precise treatment of diseases.

[0042] The double-cationic cyanine dye represented by the general formula I is described in detail below in combination with specific embodiments

[0043]

[0044] I

[0045] In the general formula I,

[0046] R1 and R2 can be independently selected from any one of methyl, ethyl, long-chain alkyl (oxy) group, benzyl, alkyl sulfonic acid group, and any one of tumor targeting groups represented by rituximab, bevacizumab, estrogen receptor, epidermal growth factor receptor, folic acid, and biotin;

[0047] X is selected from any one of dimethyl, an oxygen atom, a sulfur atom, or a selenium atom;

[0048] Y is selected from any one of iodine, chlorine, and bromine;

[0049] n can be selected from any one of 0, 1, 2, and 3;

[0050] M1 and M2 can be independently selected from one of the groups represented by the following structural formula i or ii:

[0051]

[0052] In the i and ii groups, R3 can be one of methyl, ethyl, long-chain alkyl, benzyl, and alkyl sulfonic acid group;

[0053] R4 can be selected from one of H, Cl, F, carboxylic acid group, and sulfonic acid group.

[0054] Hereinafter, specific examples of partial compounds represented by Structural Formula I will be shown, but the present application is not limited to these specific examples.

[0055]

[0056] The compounds represented by General Formula I of the present application can be synthesized by the methods described in the following examples.

[0057] Example

[0058] Example 1

[0059] The synthesis method of the dicationic cyanine dye 2LBCy5.5 includes the following steps:

[0060]

[0061] (1) Synthesis of Compound 1

[0062] A mixture of 1,4-phenylenediamine (2 g, 18.49 mmol) and 3-bromo-3-methyl-2-butanone (6.4 g, 38.78 mmol) was dissolved in chloroform (40 mL) and pyridine (10 g, 0.126 mol) under a nitrogen atmosphere. After stirring at 75°C for 3 hours, it was filtered to obtain a yellow solid (1.3 g, 4.71 mmol, 25.47%).

[0063] (2) Synthesis of Compound 2

[0064] Compound 1 (1.2 g, 4.34 mmol) was mixed with 15 mL of concentrated hydrochloric acid, and the mixture was heated to 210°C and kept at this temperature for 45 minutes, and then cooled. The cooled mixture was dissolved in water and neutralized with ammonia to obtain a yellow precipitate (0.65 g, 2.70 mmol, 62.3%).

[0065] (3) Synthesis of Compound 3

[0066] Compound 2 (2 g, 8.32 mmol) was mixed with methyl iodide (2.8 g, 11.53 mmol), and after stirring at 50°C under a nitrogen atmosphere for 18 hours, it was filtered. The remaining yellow crude product was used in the next step without further purification.

[0067] (4) Synthesis of Compound 5

[0068] Compound 4 was synthesized using 1,1,2-trimethyl-1H-benzo[e]indole. Compound 4 (4 g, 19.11 mmol), ethyl iodide (3.28 g, 21.02 mmol) were dissolved in acetonitrile (35 mL). After stirring under reflux at 110 °C for 12 h, a large amount of ethyl acetate was added to filter. The residue was a blue solid product (3.82 g, 16.04 mmol, 83.92%) which was used in the next step without further purification.

[0069] 1 H NMR (400 MHz, DMSO) δ 8.38 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.9 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.9 Hz, 1H), 7.79 (t, J = 7.0 Hz, 1H), 7.73 (t, J = 7.1 Hz, 1H), 4.62 (q, J = 7.3 Hz, 2H), 2.94 (s, 3H), 1.76 (s, 6H), 1.51 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 196.44, 138.68, 137.52, 133.52, 131.20, 130.21, 128.89, 127.76, 123.89, 113.64, 55.94, 43.76, 40.55, 21.95, 14.03, 13.33.

[0070] (5) Synthesis of compound 6

[0071] Compound 5 (3 g, 12.6 mmol) and (1E,3E)-N,N-diphenylpropane-1,3-diimine (3 g, 13.5 mmol) were dissolved in acetic acid (13 mL) and acetic anhydride (13 mL). After stirring the solvent, it was heated at 125 °C under nitrogen atmosphere for 1 h. The mixture was poured into 20 mL of methanol. The solvent was removed by rotary evaporation and the crude product was recrystallized with methyl tert-butyl ether. The product was a dark red solid (4.5 g, 10.93 mmol, 86.75%).

[0072] 1 H NMR (400 MHz, DMSO) δ 8.92 (d, J = 13.2 Hz, 1H), 8.64 (dd,J = 15.1 Hz,1H), 8.37 (d, J = 8.5 Hz, 1H), 8.22 (d, J = 8.9 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H),7.98 (d, J = 8.9 Hz, 1H), 7.76 (t, J = 7.3 Hz, 1H), 7.68 (d, 1H), 7.65 (d, 2H),7.60 (d, J = 5.1 Hz, 1H), 7.47 (d, J = 7.3 Hz, 2H), 6.94 (d, J = 15.2 Hz, 1H), 5.57(dd, J = 11.3 Hz, 1H), 4.47 (q, J = 7.0 Hz, 2H), 2.04 (s, 3H), 1.93 (s, 6H), 1.33(t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 181.34, 172.47, 156.61, 138.56,137.67, 133.19, 131.34, 130.91, 130.45, 130.09, 129.10, 128.95, 128.73,127.42, 127.12, 123.38, 119.42, 113.39, 113.14, 111.20, 53.39, 27.31, 26.13,21.54, 13.83.

[0073] (6) Synthesis of compound 2LBCy5.5

[0074] A mixture of compound 6 (0.5 g, 1.22 mmol), compound 3 (0.135 g, 0.5 mmol), dichloromethane (5 mL), triethylamine (0.5 mL), acetic anhydride (0.1 mL) was stirred at 90 °C under nitrogen atmosphere for 2 hours. The solution was poured into t-butyl methyl ether (250 mL), and a dark blue solid was obtained after filtration. The crude product was purified by silica gel column chromatography with dichloromethane / methanol (v / v, 10 / 1) as eluent, and the product was a black solid (0.165 g, 0.20 mmol, yield about 40%), named as 2LBCy5.5.

[0075] 1 H NMR (400 MHz, DMSO) δ 8.45 (t, J = 13.0 Hz, 2H), 8.34 (t, J = 13.0 Hz, 2H), 8.25 (d, J = 8.4 Hz, 2H), 8.09 (t, J = 9.5 Hz, 4H), 7.81 (s, 2H), 7.76 (d, J =8.9 Hz, 2H), 7.70 (d, J = 7.2 Hz, 3H), 7.53 (t, 2H), 6.60 (t, J = 12.2 Hz, 2H), 6.41 (d, J = 13.8 Hz, 2H), 6.27 (d, J = 13.7 Hz, 2H), 4.29 (d, J = 6.5 Hz, 4H), 3.67 (s, 6H), 1.96 (s, 12H), 1.76 (s, 12H), 1.35 (t, J = 6.8 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 173.80, 172.31, 167.46, 142.50, 140.73, 139.76, 133.79, 132.18, 132.08, 131.81, 129.13, 128.23, 128.10, 122.58, 111.91, 106.45, 104.10, 103.28, 67.88, 51.24, 49.28, 30.26, 28.83, 27.63, 27.12, 11.27. High-resolution mass spectrometry yields theoretical values ​​M[C]. 58 H 64 N4] 2+ = 408.2512, the actual high-resolution measurement value is 408.2511, see [reference]. Figure 1 .

[0076] To prove that the proportion of organic acid, organic base involved in the reaction and the selection of solvent can affect the yield of target photosensitizer, in view of the problem that the traditional synthesis method leads to low yield of the final target photosensitizer, the proportion of organic acid, organic base involved in the reaction and the selection of solvent are optimized based on the scheme of example 1, as shown in parallel experiments 1-6 in table 1.

[0077] The difference between parallel experiments 1-6 is only in step (6), see the table below for details:

[0078] Table 1 statistics of experiment optimization

[0079]

[0080] As can be seen from table 1, by statistical analysis of the average yield of multiple parallel experiments in the same batch, it is found that the yield of the product obtained according to the reaction conditions shown in parallel experiment 1 is the highest, therefore, the synthesis of photosensitizer molecules in subsequent examples is carried out according to parallel experiment 1.

[0081] Example 2

[0082] The synthesis method of dicationic cyanine dye 2LCy5.5 includes the following steps:

[0083] The difference between example 1 and this example is only that 2,3,3-trimethyl-3H-indole is used instead of 1,1,2-trimethyl-1h-benzo[e]indole, and the product is a blue-black solid with a yield of about 40%, named 2LCy5.5. The high resolution mass spectrum theoretical value is M[C 52 H 64 N4] 2+ 744.5161, and the high resolution measurement value is 372.2582, see Figure 2 .

[0084] The synthesis route involved in this example is:

[0085] .

[0086] Comparative example

[0087] Comparative example Cy5.5 is a commercial dye, the structure of which is as follows, and its maximum absorption / emission peaks are located at 691 nm and 720 nm, respectively. The product can be purchased from Bide Pharmaceutical Co., Ltd., with a purity of >99.5%.

[0088]

[0089] Cy5.5

[0090] Performance test

[0091] After drying, the compounds 2LBCy5.5, 2LCy5.5 and Cy5.5 obtained in Examples 1-2 and the comparative examples were accurately weighed and dissolved in dimethyl sulfoxide to prepare a dye mother liquor with a concentration of 2.0 mM for later use.

[0092] Test 1: Photophysical Performance Test

[0093] 20 μL of the dye stock solution was dissolved in 3.00 mL of dichloromethane solvent, and the absorption spectrum and fluorescence emission spectrum of the dye (excitation at 808 nm) were measured. The test results are shown in [reference needed]. Figure 3 .

[0094] Depend on Figure 3 It can be seen that the maximum absorption and emission peaks of the comparative molecule Cy5.5 are 691 nm and 720 nm, respectively. When it is prepared as a biscationic cyanine dye, its maximum absorption and emission peaks undergo a significant red shift, to 788 nm and 817 nm (2LCy5.5), and 808 nm and 849 nm (2LBCy5.5), respectively. Compared with Cy5.5, the absorption and emission peaks are red-shifted by 103 nm and 97 nm (2LCy5.5), and 117 nm and 129 nm (2LBCy5.5), respectively. The calculated molar extinction coefficients are 1.89 × 10⁻⁶. 5 M -1 cm -1 (2LCy5.5) and 2.09×10 5 M -1 cm -1 (2LBCy5.5), compared to Cy5.5 (1.08×10 5 M -1 cm -1 The absorption spectrum of the prepared biscation cyanine dye is approximately twice that of the original dye. The absorption spectrum ranges from 780 nm to 850 nm, and the main emission spectrum ranges from 850 nm to 900 nm. This makes it more suitable for deep tissue imaging and tumor treatment in long-wavelength absorption and emission scenarios.

[0095] Test 2 1 O2 performance test

[0096] 1,3-Diphenylisobenzofuran (DPBF) was used as... 1 O2 scavenger, utilizing DPBF's ability to react with... 1 The O2 reaction causes the color to change from yellow-green to colorless, resulting in a decrease in absorbance at the absorption peak around 412 nm. Using 808 nm and 5 mW cm⁻¹, the absorbance was adjusted. -2 Laser irradiation, using changes in DPBF fluorescence intensity to evaluate 1 O2 yield. Among them, indocyanine green was used as the reference dye molecule, and its yield in dichloromethane...1 O2yield was 7.7%.

[0097] First, 3.00 mL of methanol was added into a sealed quartz test tube, and an appropriate amount of 1,3-diphenyl isobenzofuran (DPBF) stock solution was added with a micro tube gun, mixed well, so that its absorbance at 412 nm was about 1.0, then the dye stock solution was added to adjust its absorbance to about 0.3-0.4, and 808 nm, 5 mW cm -2 Laser irradiation, DPBF absorbance was measured every 30 s, and the test results are shown in Figure 4 .

[0098] From Figure 4 It can be seen that with the extension of laser irradiation time, the absorbance of DPBF gradually decreases, indicating that it can produce reactive oxygen species for photodynamic therapy, and its 1 O2yield is calculated by the following formula (1)

[0099] 1 O2yield Φ The calculation formula is as follows:

[0100] (1)

[0101] Wherein, "sam" and "std" represent the sample to be tested and the reference sample (indocyanine green) respectively. "m" represents the slope of the decrease of DPBF absorbance at 415 nm wavelength, (O.D. is the absorbance of the sample at 808 nm).

[0102] Settlement 1 O2yield was 11.8% (2LCy5.5) and 17.9% (2LBCy5.5) respectively, which was significantly higher than that of commercial indocyanine green photosensitizer (7.7%), proving that the D-A-D type bicationic cyanine dye constructed by taking benzobispyrrole as a connecting intermediate and combining the advantages of bicationic dyes and cyanine dyes has better photosensitization efficiency (O2production capacity is stronger), that is, better photodynamic therapy effect. 1 O2yield was 11.8% (2LCy5.5) and 17.9% (2LBCy5.5) respectively, which was significantly higher than that of commercial indocyanine green photosensitizer (7.7%), proving that the D-A-D type bicationic cyanine dye constructed by taking benzobispyrrole as a connecting intermediate and combining the advantages of bicationic dyes and cyanine dyes has better photosensitization efficiency (O2production capacity is stronger), that is, better photodynamic therapy effect.

[0103] Test 3 Cell Uptake Targeting Test

[0104] MCF-7 cells were cultured in DMEM medium. 1 μM of dye stock solution was added to the culture solution containing MCF-7 cells, incubated at 37°C, and then the amount of cell uptake was determined by confocal imaging. The excitation wavelength of photosensitive molecules was 650 nm, and the emission wavelength was 700-800 nm. The test results are shown in Figure 5 .

[0105] From Figure 5It can be seen that the photosensitizing dye can quickly enter the MCF-7 cells, and it is found by re-staining with the commercial dye MTG (mitochondrial green dye) that it can be well located in the mitochondria, and the Pearson coefficients are 0.89 (2LCy5.5) and 0.82 (2LBCy5.5), respectively. The larger the Pearson coefficient, the higher the mitochondrial targeting performance. The Pearson coefficient of 2LBCy5.5 is slightly lower, mainly due to its large conjugated system and better lipophilic characteristics. In addition, it is also proved that the D-A-D type double-cationic cyanine dye constructed by taking benzobispyrrole as the connecting intermediate and organically combining the advantages of double-cationic dyes and cyanine dyes has good mitochondrial specificity, and the performance control and specific functionalization are simultaneously improved. The excitation wavelength of the photosensitizing dye is 750 nm, and the emission band is 800-850 nm. The excitation wavelength of MTG is 488 nm, and the emission band is 515-545 nm.

[0106] Test 42LBCy5.5 photosensitizing dye on MCF-7 cell light and dark toxicity experiment

[0107] In view of the more excellent photosensitization efficiency, 2LBCy5.5 is used as the target photosensitizing dye for the following cell experiments. MCF-7 cells are digested with 0.25% trypsin, and a cell suspension is prepared with DMEM culture medium containing 10% fetal bovine serum, and 10 3 ~10 4 cells are inoculated in each well of a 96-well culture plate, and the volume of each well is 100 μL; the culture plate is moved into an incubator, and incubated at 37°C, 5% CO2 and saturated humidity for 24 hours, then the same volume but different concentrations of 2LBCy5.5 photosensitizing molecules are added, and the culture is continued in the normal oxygen environment for 2 hours; then each well is irradiated with an 808 nm, 20 mW / cm 2 near-infrared light source, and after irradiation, the 96-well plate is placed in the incubator for 24 h. MTT solution (5 mg / mL) 20 μL is added to each well, incubated for 4 hours, and the culture is terminated, and the culture supernatant in the well is carefully aspirated. Then, 100 μL of DMSO is added to each well, and shaken for 10 minutes to fully dissolve the crystals; the absorbance of each well at 490 nm is measured on a microplate reader, and the cell survival rate is calculated: test group light absorbance / control group absorbance value x 100%. The test results are shown in Figure 6 .

[0108] As can be seen from Figure 6 , the 2LBCy5.5 photosensitizing dye has significant phototoxicity to MCF-7 cells under laser irradiation, and has no obvious cytotoxicity in the dark. The half lethal concentration (IC 50The singlet state and the triplet state energy gap of 2LBCy5.5 is 1.7 μM. Due to the regulation of the structure, the singlet state and the triplet state energy gap is reduced, which is conducive to promote intersystem crossing process, resulting in more singlet oxygen, 2LBCy5.5 can be used for anti-tumor photodynamic therapy.

[0109] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, but not limited to them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A biscationic cyanine dye, characterized in that, The dicationic cyanine dye has the following general structural formula I: General Formula I In general formula I: R1 and R2 can each be independently selected from either methyl or ethyl; X is selected from any one of dimethyl, oxygen, sulfur or selenium atoms; Y is selected from any one of iodine, chlorine, and bromine; n is 1; M1 and M2 may be the same or different, and each of M1 and M2 is independently selected from the following groups: R3 can be either methyl or ethyl.

2. The biscationic cyanine dye according to claim 1, characterized in that, The absorption and emission wavelengths of the biscationic cyanine dye in the near-infrared I region are 750 ~ 900 nm; the molar extinction coefficient of the biscationic cyanine dye in the near-infrared I region is 1.88 ~ 2.1 × 10⁻⁶. 5 M -1 cm -1 .

3. A method for preparing a biscationic cyanine dye according to any one of claims 1-2, characterized in that, Includes the following steps: Using compounds II and III as raw materials, a Knoevenagel condensation reaction occurs in organic acid anhydrides, organic bases, and nonpolar solvents to generate near-infrared dicationic cyanine dyes; In compound III, M is selected from M1 or M2.

4. The preparation method according to claim 3, characterized in that, The molar ratio of compound II to compound III is 1:1 to 3, the molar ratio of compound II to organic base is 1:4 to 5, and the molar ratio of compound II to organic anhydride is 1:1.5 to 3.

5. The preparation method according to claim 3, characterized in that, The organic acid anhydride is selected from any one of acetic anhydride, propionic anhydride, succinic anhydride, benzoic anhydride, and phthalic anhydride.

6. The preparation method according to claim 3, characterized in that, The organic base is selected from triethylamine, pyridine, and N,N-diisopropylethylamine.

7. The preparation method according to claim 3, characterized in that, The nonpolar solvent is selected from one of dichloromethane, trichloromethane, and dichloroethane.

8. The biscationic cyanine dye according to any one of claims 1-2 is used in bioimaging and pharmaceutical phototherapy formulations.

9. The application according to claim 8, characterized in that, The biscationic cyanine dyes are used in formulations for normoxic and hypoxic photodynamic therapy of tumors.

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