A selenocyanine compound and its application

Abstract

The present invention discloses a class of selenocyanine compounds having the following structure: wherein R represents -OH, -NH2 or N,N-diethylamino; X ‑ Represents anion. The absorption and emission wavelengths of the selenocyanine compounds of the present invention are both located in the near-infrared region (700nm-900nm). They effectively target mitochondria and can be used as fluorescent dyes for cell and in vivo imaging and tumor-targeted photodynamic / photothermal combined therapy.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a novel selenocyanine compound and an application thereof as a near-infrared emitting fluorescent dye. Background Art

[0002] In recent years, phototherapy has attracted the research interest of researchers as a non-invasive, real-time tumor treatment method. Compared with traditional chemotherapy and radiotherapy, photodynamic therapy (PDT) and photothermal therapy (PTT) have attracted widespread attention due to their superior spatial specificity, non-invasiveness and low side effects on normal tissues. PDT refers to the process in which photosensitizers (PSs) absorb the energy of lasers of specific wavelengths and transfer the energy to surrounding oxygen molecules, generating chemically active reactive oxygen species (ROS), especially singlet oxygen ( 1O2), thereby killing cancer cells. PTT refers to the absorption of near-infrared light energy by photothermal agents and converting it into heat energy, causing thermal damage and thermal ablation to tumor cells or tissues. However, the therapeutic effect of single therapy is limited by the complex tumor microenvironment, resulting in the inability to effectively and completely eliminate the tumor. Therefore, PTT is often combined with PDT to improve the effect of tumor treatment. In combined therapy, the heat generated by PTT can not only promote blood flow and increase oxygen content in the tumor, but also enhance the cell uptake of photosensitizers (PSs), thereby significantly improving its anti-cancer effect. (Wei,X.; Zhang C.; HeS.S.; Huang JG; Huang,JS; Liew,SS; Zeng,ZL; Pu,KY. therapy.Angew.Chem.Int.Ed.2022,e202202966; Feng, GX; Zhang, GQ; Ding, D., Design of superior phototheranostic agents guided by Jablonski diagrams.Chem.Soc.Rev.2020,49,8179-8234;

[0003] Fluorescence imaging is an emerging imaging technology with the advantages of simple operation, high sensitivity, and little harm to organisms. It has great advantages in biomedical research and has been applied to research fields such as drug action mechanism, oncology, and in vivo fluorescence imaging. In particular, near-infrared fluorescence (NIR) imaging uses excitation light and emission light with longer wavelengths and lower energy. Fluorescence imaging in the near-infrared region (650nm~900nm) can effectively avoid the self-absorption and autofluorescence interference of biological tissues, becoming the "optical window" for in vivo imaging. Due to their good biocompatibility, excellent photophysical properties, and highly diverse structural tunability, merocyanine dyes are often used as near-infrared fluorescent reporter molecules for imaging applications in targeted anticancer drug delivery monitoring and detection of various biological species. Unfortunately, most merocyanines have singlet oxygen ( 1 O2) efficiency (Φ Δ) is still unsatisfactory. A commonly used strategy is to incorporate heavy atoms (such as Br, I, Pt, Ir, and Ru) into merocyanines to improve intersystem crossing (ISC) efficiency and ROS generation. For example, Tan Weihong's group developed a mitochondrial-targeted prodrug PNPS, whose structure contains a brominated oxyanthracene merocyanine photosensitizer and an anticancer drug 5'-deoxy-5-fluorouridine for synergistic chemotherapy-photodynamic therapy. Peng Xiaojun's group reported a class of iodinated oxyanthracene merocyanine hypoxia probes ICy-N for precise tumor hypoxia imaging and inhibition of tumor growth through the PDT process.(Liu, HW; Hu, XX; Li, K.; Liu, YC; Rong, QM; Zhu, LM; Yuan, L.; Qu, FL; Zhang, XB; Tan, WH Chem. Science Pigments,2021,184,108854;Santra,M.;Owens,M.;Birch,G.;Bradley,M.,Near-infrared-emittinghemicyanines and their photodynamic killing of cancer cells.ACS Appl.BioMater.2021,4,8503-8508;Xu,F.;Li,HD;Yao,QC;Ge,HY;Fan,JL;Sun,W.;Wang,JY;Peng,XJ,Hypoxia-activated NIR photosensitizer anchoring in themitochondria for photodynamic therapy.Chem.Sci.2019,10,10586-10594)However, the addition of heavy atoms to the original mitochondria structure undoubtedly increases the difficulty of synthesis, and the introduction of heavy atoms increases the dark toxicity of cells and the limited absorption and emission wavelengths seriously hinder the feasibility of phototherapy for tumors. In addition, in the process of returning from the excited state to the ground state, fluorescence imaging and phototherapy strategies PDT / PTT are related to energy radiation and non-radiation processes, respectively, which are almost opposite to each other and always competitive. Therefore, it is difficult to enhance both at the same time in a single chromophore. At the same time, it is still a great challenge to avoid unforeseen photodegradation and bleaching while expanding the imaging window of merocyanine dyes and improving their therapeutic effects. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a novel selenocyanine compound and application thereof.

[0005] The specific technical solutions of the present invention are as follows:

[0006] A selenocyanine compound having the following structure:

[0007] Wherein R represents -OH, -NH2 or N,N-diethylamino;

[0008] X - Represents anions, such as Cl - Br - ,I - 、NO3 - or PF4 - .

[0009] The present invention further discloses a polyethylene glycol-modified selenocyanine compound, which is characterized by having the following structure: Wherein R represents -OH, -NH2 or N,N-diethylamino; X - Represents anions, such as Cl - Br - ,I - 、NO3 - or PF4 - ; n = an integer from 3 to 200.

[0010] In a specific example of the present invention, R represents N,N-diethylamino, and the polyethylene glycol is PEG5000 (n=79).

[0011] The absorption and emission wavelengths of the selenocyanine compounds and polyethylene glycol-modified selenocyanine compounds described in the present invention are both located in the near-infrared region (700-900 nanometers). Another object of the present invention is to protect the use of the above compounds in the preparation of fluorescent dyes or tumor phototherapy drugs. Furthermore, the fluorescent dye is a fluorescent probe for cell and in vivo imaging.

[0012] Another object of the present invention is to provide a method for synthesizing the selenocyanine compound and the selenocyanine compound modified with polyethylene glycol. According to the strong nucleophilicity of selenophene, 3-diethylaminobenzelenophene and Sichuan cyanine dye IR780 are selected for one-step reaction to obtain the selenocyanine compound CySe-NEt2. CySe-Alkynyl and N3-mPEG-Methoxy are reacted by copper (I)-catalyzed alkyne-azide click reaction to obtain the selenocyanine compound modified with polyethylene glycol. The synthesis method of the present invention is simple, the synthesis conditions are mild, and the purification is relatively simple.

[0013] Beneficial Effects

[0014] (1) Compared with oxygen / thiomerocyanine dyes (Yuan, L.; Lin, WY; Zhao, S.; Gao, WS; Chen, B.; He, LW; Zhu, SS, A unique approach to development of near-infrared fluorescent sensors for in vivo imaging. J. Am. Chem. Soc., 2012, 134, 13510-13523), the absorption wavelength of the selenocyanine compound of the present invention is red-shifted to 776 nm, the emission wavelength is red-shifted to 818 nm, the singlet oxygen yield is increased to 14.8%, and the photothermal conversion efficiency is enhanced to 42.2%. The dye targets cell mitochondria and produces more reactive oxygen species (ROS) under light to kill cells. It also has obvious phototoxicity, and its half-lethal concentration IC50 value reaches 0.26 μM, which is a potential effective photosensitizer.

[0015] (2) The present invention modifies the structure of selenocyanine compounds with polyethylene glycol to obtain CySe-mPEG 5K The results showed that CySe-mPEG 5K It not only inherits the photothermal conversion ability of CySe-NEt2, but also has a singlet oxygen yield of 14.8% and a photothermal conversion efficiency of 14.2%. More importantly, compared with the poor photostability of CySe-NEt2, it has high photothermal conversion ability and stability. 5K It can effectively enrich in the tumor area, realize the imaging of the tumor area under the photoacoustic mode, and provide more accurate spatiotemporal information for clinical treatment of tumors. It has broad application prospects in biology and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . Normalized absorption spectra (a) and fluorescence spectra (b) of different oxygen-containing cyanine compounds CyX-NEt2 (X=O, S or Se).

[0017] Figure 2 . Singlet oxygen yield and photothermal effect test diagram of CyX-NEt2. Changes in absorption spectrum under illumination conditions, with an interval of 5 seconds. (a)-(c) Spectral changes of DPBF and CyX-NEt2 in methanol solution after laser illumination, with an excitation wavelength of 750nm and an irradiation power of 20mW / cm 2; (d) The change of DPBF absorption value at 410nm caused by irradiation of ICG and CyX-NEt2 over time. (e) The change of temperature of CyX-NEt2 (50μM) and PBS (pH=7.4) over time under 750nm laser irradiation. When the temperature reaches the maximum value, the irradiation is stopped and the temperature change curve is recorded by infrared thermal imager; (f) The hot-cold curve of CySe-NEt2, a total of 5 cycles, the laser wavelength is 750nm, and the optical power density is 500mW / cm 2 .

[0018] Figure 3 . False color images of 4T1 cell fluorescence confocal imaging of CyX-NEt2. (a) CyO-NEt2; (b) CyS-NEt2; (c) CySe-NEt2 co-localization imaging. Dye CyX-NEt2 channel, 670nm excitation, 680nm-800nm ​​channel collection; mitochondrial dye MitoTracker Green and lysosomal dye LysoTracker Green channel, 488nm excitation, 492nm-630nm channel collection; cell nuclear dye DAPI channel, 405nm excitation, 420nm-450nm channel collection. Bar value: 25μm.

[0019] Figure 4 . is the phototherapy effect of CyX-NEt2 on 4T1 cells. (a) Intracellular ROS detection experiment, cells were incubated with CyX-NEt2 (4μM) and then protected from light or irradiated with light. DCFH-DA (1μM) is an indicator for intracellular reactive oxygen species detection. Green fluorescence channel, 488nm excitation, 500nm-550nm channel collection, Bar value: 20μm; (b) is the bar graph of the average fluorescence intensity in the cells corresponding to (a), n=3; (c) The survival rate of 4T1 cells incubated with different concentrations (0-4μM) of CyX-NEt2 after 24 hours of protection from light or irradiation, the laser wavelength is 750nm, and the light power density is 500mW / cm 2 (d) Live and dead cell staining of 4T1 cells after incubation with CyX-NEt2 (4 μM) and illumination, live cell staining agent Calcein AM, dead cell staining agent Propidium Iodide (PI), green channel, 488 nm excitation, 500 nm-550 nm channel collection; red channel, 543 nm excitation, 560 nm-650 nm channel collection. Bar value: 25 μm.

[0020] Figure 5 . The polyethylene glycol-modified selenocyanine compound CySe-mPEG of the present invention 5KPhotothermal effect of CySe-mPEG under 750nm laser irradiation 5K The temperature change of PBS (pH=7.4) solution (50 μM) changes with time. When the temperature reaches the maximum value, the light is stopped and the temperature change curve is recorded by infrared thermal imager; (b) CySe-mPEG 5K Hot-cold curve, 5 cycles in total, laser wavelength 750nm, optical power density 500mW / cm 2 ; (c) at 750nm laser, 500mW / cm 2 Under light power density irradiation, CySe-mPEG with different concentrations (20μM, 40μM and 60μM) 5K Infrared temperature change curve; (d) CySe-mPEG 5K The concentration was 60 μM and the power (200 mW / cm 2 , 300mW / cm 2 and 500mW / cm 2 )Infrared temperature change curve under .

[0021] Figure 6 . The polyethylene glycol-modified selenocyanine compound CySe-mPEG of the present invention 5K (a) Incubation with different concentrations (0-64 μM) of CyX-NEt2 and CySe-mPEG 5K (a) The survival rate of 4T1 cells after being protected from light for 24 hours; (b) Incubation with different concentrations (0-4 μM) of CySe-mPEG 5K The survival rate of 4T1 cells after 24 hours of light treatment, the laser wavelength is 750nm, the light power density is 500mW / cm 2 .

[0022] Figure 7 . The polyethylene glycol-modified selenocyanine compound CySe-mPEG of the present invention 5K Tumor-targeted fluorescence / photoacoustic imaging. (a) Tail vein administration, CySe-NEt2 and CySe-mPEG 5K (50μM) fluorescence angiography image in 4T1 tumor-bearing mice; (b) fluorescence angiography of tumor tissue and normal tissue 1 hour, 2 hours and 3 hours after administration, excitation wavelength 780nm, collection band 845±20nm; (c) is the fluorescence intensity of the tumor area in (a); (d) is the fluorescence intensity of the tumor tissue and normal tissue in (b) at different time periods of CySe-NEt2; (e) is the fluorescence intensity of the CySe-mPEG in (b) 5K Fluorescence intensity of tumor tissue and normal tissue at different time periods; (f) CySe-NEt2 and CySe-mPEG5K Photoacoustic imaging images of the tumor area every 1 hour; (g) is the photoacoustic signal PA of the tumor area in (f) 780 Strength value, scale: 3mm.

[0023] Figure 8 . The polyethylene glycol-modified selenocyanine compound CySe-mPEG of the present invention 5K Tumor photothermal imaging. (a) PBS+780nm illumination group, CySe-NEt2+780nm illumination group and CySe-mPEG 5K Thermal imaging of mice in the +780nm light irradiation group; (b) is the temperature rise curve of the tumor site in (a).

[0024] Fig. 9 . The polyethylene glycol-modified selenocyanine compound CySe-mPEG of the present invention 5K Evaluation of tumor phototherapy. (a) Weight change curve of tumor-bearing mice in different experimental groups during treatment; (b) Tumor volume growth curve of mice in different experimental groups; (c) Photos of body size and tumor sites of tumor-bearing mice in different experimental groups after the end of the experiment; (d) Photos of in vitro tumors in different experimental groups after two weeks of treatment; (e) Hematoxylin-eosin stained sections of the main organs and tissues of mice in different experimental groups, scale: 100μm.

[0025] Specific implementation methods The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to the following examples.

[0026] Example 1: Preparation of different oxygen-containing merocyanine compounds CyX-NEt2 (X = O, S or Se)

[0027] 1. Preparation of CyO-NEt2

[0028]

[0029] Synthesis of compound CyO-NEt2: Dissolve CyCHO-NEt2 (280 mg, 1.0 mmol), CH3COONa (164 mg, 2.0 mmol) and indole salt (395 g, 1.2 mmol) in anhydrous Ac2O (25 mL) and stir at 25°C for 18 hours. Concentrate the obtained dark green solution under reduced pressure and dissolve the obtained residue in dichloromethane, then wash with water 3 times, and dry the organic layer with anhydrous sodium sulfate. Evaporate the solvent and purify with silica gel column, eluent: CH2Cl2:CH3OH=100:3, to obtain 0.38 g of dark green solid, with a yield of about 65%. 1H NMR (400MHz, CD2Cl2, δppm): 8.54 (d, J=14.2Hz, 1H), 7.53-7.40 (m, 4H), 7.30 (td, J=7.5, 0.7 Hz,1H),7.21(d,J=7.9Hz,1H),6.84(dd,J=9.0,2.4Hz,1H),6.55(d,J=2.0Hz,1H),6.08(d,J= 14.2Hz,1H),4.07(t,J=7.5Hz,2H),3.55(q,J=7.1Hz,4H),2.78(t,J=6.1Hz,2H),2.69(t,J= 6.1Hz,2H),1.99-1.86(m,4H),1.77(s,6H),1.29(t,J=7.1Hz,6H),1.09(t,J=7.4Hz,3H)ppm. 13 C NMR (400MHz, CD2Cl2): δ174.69,165.12,157.78,153.62,143.64,143.45,142.16,139.94,130.94,130.06,126.67,124.63,123.67,116.3 8,114.51,113.79,112.22,100.72,97.02,55.21,50.78,47.49,46.58,30.04,29.63,25.81,22.03,13.59,12.71ppm.HR-MS(ESI,positive mode,m / z):calcd.467.30569,found467.30423for[MI] + .

[0030] 2. Preparation of CyS-NEt2

[0031]

[0032] Synthesis of compound CyS-NEt2: Under N2 atmosphere and 0°C, compound CyS-NH2 (200 mg, 0.361 mmol) was dissolved in CH3CN (30 mL) solution, iodoethane (281 mg, 1.8 mmol) and K2CO3 (200 mg, 1.44 mmol) were added, and refluxed at 85°C for 18 h. The reaction solvent was removed under reduced pressure, diluted with dichloromethane, and then washed with water 3 times, and the organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated and purified by silica gel chromatography, eluent: CH2Cl2:CH3OH=50:1, and a blue-green solid yield of about 15% was obtained. 1H NMR (400MHz, CD2Cl2, δppm): 8.24 (d, J=13.6Hz, 1H), 7.51 (dd, J=14.8, 8.1Hz, 2H), 7.43 (dd, J=1 6.9,9.2Hz,2H),7.32(t,J=7.4Hz,1H),7.21(d,J=7.9Hz,1H),6.92(d,J=10.5Hz,2H),6.17(d,J= 13.7Hz,1H),4.06(t,J=7.3Hz,2H),3.53(dd,J=13.9,6.9Hz,4H),2.79(t,J=5.8Hz,2H),2.67(t ,J=5.8Hz,2H),2.06-1.87(m,4H),1.79(s,6H),1.28(t,J=6.9Hz,6H),1.07(t,J=7.3Hz,3H)ppm. 13 C NMR (400MHz, CD2Cl2): δ174.67,159.09,151.10,143.29,142.46,141.96,140.66,134.71,131.68,130.12,126.90,126.58,123.78,120.7 5,115.91,112.34,106.16,101.97,97.33,50.91,47.50,46.45,33.66,29.94,28.03,22.10,22.04,13.68,12.70ppm.HR-MS(ESI,positive mode,m / z):calcd.483.28284,found483.28107for[MI] + .

[0033] 3. Preparation of CySe-NEt2 of the present invention

[0034]

[0035] Synthesis of compound CySe-NEt2: NaBH4 (23 mg, 0.6 mmol) was added in batches to a solution of 2ArSe-NEt2 (91 mg, 0.2 mmol) in EtOH (2 mL) under N2 atmosphere at 0°C. After 15 minutes of reaction, solid citric acid (192 mg, 1.0 mmol) was added and the reaction mixture was stirred at 0°C for 5 minutes. The reaction solution was then diluted with Et2O (5 mL) and deionized water (3 mL) was added. After extraction, the layers were separated and the organic layer was washed with saturated NH4Cl aqueous solution (2 mL) and brine (2 mL), dried over Na2SO4, filtered and concentrated in vacuo to obtain ArSeH-NEt2 without further purification.1 H NMR (400MHz, CD2Cl2, δppm): 7.09 (t, J = 7.9Hz, 1H), 6.97-6.88 (m, 2H), 6.54 (d, J = 7.5Hz, 1H), 3.30 (q, J = 7.0Hz, 4H), 1.12 (t, J = 7.1Hz, 6H) ppm.

[0036] Compound ArSeH-NEt2 (361 mg, 1.58 mmol) and K2CO3 (218 mg, 1.58 mmol) were dissolved in acetonitrile solution (20 mL), heated at 50°C and stirred in an Ar atmosphere for 10 min. IR780 (700 mg, 1.05 mmol) was dissolved in acetonitrile (50 mL), slowly added dropwise to the above solution with a syringe, reacted at room temperature in the dark for 24 h, and followed by TCL. The solvent was evaporated and purified by silica gel chromatography, the developing solvent was CH2Cl2:CH3OH=94:4, and a blue solid was obtained with a yield of about 20%. 1 H NMR (400MHz, CD2Cl2, δppm): 8.08 (d, J = 13.7Hz, 1H), 7.53 (d, J = 8.6Hz, 2H), 7.46 (t, J = 7.6Hz, 1H ),7.36(t,J=7.4Hz,1H),7.30-7.24(m,2H),7.04(d,J=1.9Hz,1H),6.85(d,J=9.0Hz,1H),6.30( d,J=13.7Hz,1H),4.12(t,J=7.4Hz,2H),3.51(q,J=7.0Hz,4H),2.81(t,J=6.0Hz,2H)),2.66(t, J=6.0Hz,2H),2.03-1.84(m,4H),1.79(s,6H),1.27(t,J=7.0Hz,6H),1.07(t,J=7.3Hz,3H)ppm. 13 C NMR (400MHz, CD2Cl2): δ174.36,161.53,149.46,144.40,142.03,141.59,141.36,139.08,134.76,130.56,128.92,128.75,126.09,122.57 ,120.03,113.91,111.47,107.59,102.26,49.95,46.52,45.07,33.43,28.51,27.56,21.03,20.91,12.37,11.37ppm.HR-MS(ESI,positive mode,m / z):calcd.531.22730,found 531.22687for[MI]+ .

[0037] Normalized absorption (a) and fluorescence emission (b) spectra of CyX-NEt2 in pure methanol solvent Figure 1 shown.

[0038] The maximum absorption wavelength of CyO-NEt2 is around 716 nm, and the corresponding maximum molar extinction coefficient is 1.37×10 5 M -1 cm -1 The maximum fluorescence emission wavelength is 746nm, and the fluorescence quantum yield is 0.23. The maximum absorption and emission wavelengths of the compound CyS-NEt2 are 760nm and 800nm, respectively, the fluorescence quantum efficiency is 0.18, and the molar extinction coefficient is 1.17×10 5 M -1 cm -1 The maximum absorption and emission wavelengths of the compound CySe-NEt2 are 776nm and 818nm, respectively, the fluorescence quantum efficiency is 0.11, and the molar extinction coefficient is 1.08×10 5 M -1 cm -1 By comparison, it can be seen that the absorption and emission wavelengths of selenocyanines are longer than those of oxo / thiocyanines, which is more conducive to near-infrared imaging. Its relatively low fluorescence quantum yield means that non-radiative energy dissipation is greater, which is more conducive to singlet oxygen generation and photothermal conversion.

[0039] Example 2: Polyethylene glycol-modified selenocyanine compound CySe-mPEG 5K Preparation

[0040]

[0041] Compound ArSeH-NEt2 (80 mg, 0.35 mmol) and K2CO3 (48 mg, 0.35 mmol) were dissolved in acetonitrile solution (20 mL), heated at 50°C and stirred in an Ar atmosphere for 10 min. IR780-Alkynyl (166 mg, 0.23 mmol) was dissolved in acetonitrile (50 mL), slowly added dropwise to the above solution with a syringe, and reacted at room temperature in the dark for 24 h, and the reaction was tracked by TCL. The solvent was evaporated and purified by silica gel chromatography, the developing solvent was CH2Cl2:CH3OH=94:4, and a blue solid was obtained with a yield of about 25%. 1H NMR (400MHz, CD2Cl2, δppm): 8.07 (d, J = 13.6Hz, 1H), 7.53 (dd, J = 12.0, 8.5Hz, 2H), 7.45 (d, J = 7.5Hz, 1 H),7.36(dd,J=16.4,8.0Hz,2H),7.28(s,1H),7.01(s,1H),6.86(d,J=8.6Hz,1H),6.45(d,J=13.6Hz,1 H),4.34(t,J=7.1Hz,2H),3.51(dd,J=13.7,6.7Hz,4H),2.80(d,J=4.7Hz,2H),2.69(d,J=5.4Hz,2H),2 .44(s,2H),2.22(s,1H),2.13-2.04(m,2H),1.98-1.92(m,2H),1.78(s,6H),1.27(t,J=6.8Hz,6H)ppm. 13 C NMR (400MHz, CD2Cl2, δppm):174.56,162.10,149.90,144.94,144.78,142 .24,141.67,139.70,135.28,131.16,129.55,129.38,126.40,122.90,120 .52,114.41,111.86,107.90,102.71,83.21,70.47,50.24,45.48,43.93,33.84,28.91,27.98,26.49,21.42,16.38,12.77ppm. HR-MS (ESI, positive mode,m / z):calcd.555.22730,found555.22601for[MI] + .

[0042] CySe-mPEG 5K It was obtained by copper (I) catalyzed alkyne-azide click reaction. CySe-Alkynyl (20 mg, 0.029 mmol), CuSO4·5H2O (7.33 mg, 0.029 mmol), sodium ascorbate (11.62 mg, 0.058 mmol) and N3-mPEG-Methoxy (M n =5000) (155 mg, 0.029 mmol) was dissolved in DMSO / H2O (5 / 5 mL). The reaction mixture was stirred overnight at room temperature under nitrogen atmosphere, and then the reaction solution was dialyzed with deionized water for 24 hours to remove excess salt, N3-mPEG-Methoxy and DMSO, and freeze-dried to obtain CySe-mPEG5K , with a yield of 95%. 1 HNMR (400MHz, MeOD, δppm): 8.18 (d, J = 13.3Hz, 1H), 7.69-7.61 (m, 3H), 7.57-7.48 (m, 2H), 7.45- 7.39(m,2H),7.26(s,1H),6.99(d,J=7.8Hz,1H),6.49(d,J=13.7Hz,1H),4.62(s,2H),4.38(s,2H ),4.15(s,2H),4.06-3.54(m,mPEG),3.50(dd,J=9.3,4.5Hz,4H),3.40(s,3H),3.27(d,J=5.0Hz, 2H), 2.87 (s, 2H), 2.70 (s, 2H), 2.33 (s, 2H), 2.01 (s, 2H), 1.82 (s, 6H), 1.30 (t, J = 6.9Hz, 6H) ppm. Modi-TOF-MS, m / z: found 5000~6000.

[0043] Example 3: Photodynamic and photothermal effects of different oxygen-containing merocyanine compounds CyX-NEt2 (X = O, S or Se)

[0044] The singlet oxygen generation ability of the fluorescent dye CyX-NEt2 was investigated. 1,3-Diphenylisobenzofuran (DPBF) was selected as the scavenger of singlet oxygen. DPBF itself has an absorption peak at 415nm. When singlet oxygen is present, DPBF reacts with singlet oxygen, destroying its conjugated structure, causing the ultraviolet absorption peak at 410nm to decrease. The reaction process is shown below:

[0045]

[0046] Indocyanine Green (ICG) with matching wavelength was selected as the standard singlet oxygen reference, and DPBF was used as the singlet oxygen scavenger. The results were as follows: Figure 2 As shown. Figure 2 As can be seen from (a)-(d), in methanol solvent, the wavelength is 750nm and the power is 5mW / cm 2After 1 minute of laser irradiation, the fluorescent dye CySe-NEt2 rapidly produces singlet oxygen after illumination, which is manifested by a rapid decrease in the DPBF absorption peak at 410nm, while the DPBF absorption peak in the CyS-NEt2 solution decreases slightly, and there is almost no change in CyO-NEt2. According to the following formula, the singlet oxygen yields of the compounds CyO-NEt2, CyS-NEt2 and CySe-NEt2 are 0.9%, 2.5% and 14.8%, respectively. The results show that CySe-NEt2 is more suitable for photodynamic therapy than CyO-NEt2 and CyS-NEt2.

[0047]

[0048] φ Δ represents the singlet oxygen yield; the superscripts sample and ICG represent the material and the reference substance, respectively; S represents the slope of the plot of the change in the absorbance value of DPBF at 410 nm versus time; F = 1-10 -OD , OD value is the absorbance of material and ICG at 750nm.

[0049] Photothermal effect: Use 750nm excitation light source (0.5W·cm -2 ) 50 μM CyX-NEt2 in PBS buffer was illuminated for 150 seconds, then the laser was turned off and allowed to cool to the initial temperature, and the temperature was recorded once per second. Figure 2 In (e), it can be seen that when the initial solution temperature is 25°C, after laser irradiation, the solution temperature can rise to 41°C (CyO-NEt2), 53°C (CyS-NEt2) and 63°C (CySe-NEt2), respectively, while the temperature of pure PBS buffer solution has no obvious change. Through calculation, it is known that the thermal conversion efficiency is 27.5% (CyO-NEt2), 38.5% (CyS-NEt2), and 42.2% (CySe-NEt2), respectively, indicating that CySe-NEt2 has more advantages in tumor photothermal therapy.

[0050] Photothermal stability test, that is, repeatedly measuring the heating / cooling process under a certain light power density, and determining the photothermal stability of the material by the change in temperature each time under the same conditions. Figure 2 As shown in (f), the results show that the temperature of CySe-NEt2 cannot continue to rise after 5 cycles, which may be due to the destruction of the conjugated structure of the merocyanine by thermal effects and singlet oxygen.

[0051] Example 4: Colocalization experiment of different oxygen-containing cyanine compounds CyX-NEt2 (X = O, S or Se) The 4T1 cells used in the imaging experiment were cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 10% embryonic calf serum (FBS, Invitrogen), penicillin (100 units / mL) and streptomycin (100 mg / mL) at 37°C and 5% CO2 in air.

[0052] 4T1 cells were incubated with 2μM CyX-NEt2 at 37°C for 30 minutes and then washed three times with PBS (20mM, pH 7.4). Then, they were incubated with 150nM co-staining reagent (including Mito-Tracker Green, Lyso-Tracker Green and DAPI) for 30 minutes, and then washed three times with PBS (20mM, pH 7.4) for imaging. Red channel excitation wavelength: 670nm; emission wavelength range: 680-800nm. Green channel excitation wavelength: 488nm; emission wavelength range, 492-630nm. Blue channel excitation wavelength: 405nm; emission wavelength range, 420-450nm. Co-localization experiments were photographed on a Leica TCS SP8 laser confocal fluorescence microscope using a 63x oil objective. Co-staining reagents were purchased from Thermo Fisher Scientific, and the experiments were performed according to the instructions. Figure 3 It can be seen that after 30 minutes of co-incubation of the mitochondrial commercial dye Mito-tracker Green and CyX-NEt2, the fluorescence of the two can overlap well, and the red and green fluorescence appear yellow after superposition. The Pearson's colocalization coefficients are 0.89 (CyO-NEt2), 0.89 (CyS-NEt2) and 0.91 (CySe-NEt2), respectively, while the Pearson's colocalization coefficients of Lyso-Tracker Green and DAPI after co-incubation are less than 0.7, indicating that the CyX-NEt2 dye targets mitochondria. The above results may be due to the negative mitochondrial membrane potential, and the positively charged merocyanine structure is conducive to targeting mitochondria.

[0053] Example 5: Cell phototherapy effects of different oxygen-containing merocyanine compounds CyX-NEt2 (X = O, S or Se)

[0054] Intracellular ROS detection experiment: The commercial reactive oxygen detection kit DCFH-DA (2′,7′-dichlorofluorescein diacetate) was selected as the indicator for intracellular reactive oxygen detection, and 4T1 cells were used as the object of investigation. DCFH-DA itself does not emit fluorescence. After being taken up by 4T1 cells, the esterase in the cells can hydrolyze it into DCFH. DCFH cannot penetrate the cell membrane, so the probe is easily labeled in the cell. When reactive oxygen exists in the cell, DCFH will be oxidized into the fluorescent substance DCF. Under 488nm light excitation, DCF will emit fluorescence at 525nm, and its fluorescence intensity is proportional to the concentration of reactive oxygen. After incubating 4T1 cells with a concentrated CyX-NEt2 solution or PBS (pH=7.4) at 37°C in air containing 5% CO2 for 2h, the excess material was washed away with PBS buffer. After adding DCFH-DA, 30 minutes later, it was irradiated with a 750nm laser for 5 minutes, and then used for imaging experiments. By Figure 4 In (a), it can be seen that the cells in the CyO-NEt2 and CyS-NEt2 groups showed weak green fluorescence, while the cells in the CySe-NEt2 group showed strong green fluorescence. Figure 4 Middle (b) shows that compared with cells in other groups, a large amount of singlet oxygen was produced in the cells of the CySe-NEt2 group.

[0055] MTT assay: The dark toxicity and phototoxicity of compound CyX-NEt2 on 4T1 cells were studied using the MTT assay. 4T1 cells were seeded into 96-well plates at a seeding density of approximately 1×10 5 After inoculation, continue to culture in the incubator for 24 hours. After the cells adhere to the wall, aspirate the culture medium in the well and add different concentrations of 4T1 cells. Continue to culture for 6 hours and then use 20mW / cm 2 The cells were illuminated with a 750nm laser of high energy for 5 minutes. After the illumination, the cells were cultured for 24 hours. Then, 40μL of 2.5mg / mL MTT solution was added to each well. After 4 hours of incubation, the solution in the wells was aspirated, 150μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, and the absorbance OD value of each well at 490nm was measured using an ELISA reader (Thermo Scientific, Varioskan Flash). The cell growth survival rate of the control group without drug addition was 100%, and the calculation formula was: Cell survival rate % = OD of drug addition group / OD of control group × 100%.

[0056] The test results show that if Figure 4 In (c), compound CyX-NEt2 (X = O, S, Se) was incubated with cells for 24 hours in the dark, and the half inhibitory concentration of cells was IC 50were 12.08μM, 8.02μM and 5.33μM, while under light conditions, the cell survival rate was significantly reduced, and the half inhibitory concentration IC 50 >4μM, 2.17μM and 0.26μM, respectively, and the corresponding PI values ​​(IC 50(Light) / IC 50(Dark) ) were <3.02, 3.70 and 20.50, respectively, which proved that compared with CyO-NEt2 and CyS-NEt2, compound CySe-NEt2 produced obvious phototoxicity and had better photodynamic therapy effect.

[0057] Live and dead cell staining experiment: CySe-NEt2 solution was incubated with 4T1 cells at 37°C for 4 hours, and then the excess material was washed with PBS buffer. Calcein AM and propidium iodide (PI), stains for live and dead cells, were then added, and the cells were irradiated with a 750nm laser for 5 minutes before imaging. Figure 4 As can be seen in (d), when there is no illumination, the cells show green fluorescence, but after illumination, the cells show red fluorescence, and the cell morphology changes significantly, which once again proves that CySe-NEt2 has a good phototherapy effect.

[0058] Example 6: Polyethylene glycol-modified selenocyanine compound CySe-mPEG 5K Photothermal effect

[0059] Referring to the method of Example 3, CySe-mPEG 5K The photothermal effect of Figure 5 As shown in (a) and (b). 5K The thermal conversion efficiency of CySe-mPEG is 40.5%, indicating that 5K CySe-mPEG inherits the photothermal conversion ability of CySe-NEt2. More importantly, compared with the poor photostability of CySe-NEt2, CySe-mPEG 5K Under 730nm laser irradiation, CySe-mPEG 5K The photothermal conversion ability of CySe-mPEG was almost unchanged. 5K It has high photothermal conversion ability and stability, which is superior to CySe-NEt2.

[0060] Concentration / light power density dependent photothermal conversion test: Figure 5 As shown in (c), CySe-mPEG is fixed 5K The concentration of CySe-mPEG was 50 μM. 5KThe temperature change in PBS shows a positive correlation with the optical power density. For example, when the laser power density is 200mW / cm 2 ,300mW / cm 2 and 500mW / cm 2 When the temperature rises to 33℃, 40℃ and 60℃, the Figure 5 As shown in (d), the laser power density is fixed at 500mW / cm 2 When CySe-mPEG 5K With increasing concentrations, CySe-mPEG 5K The temperature change in PBS also increases accordingly. When the concentrations are 20μM, 40μM and 60μM, the corresponding temperatures rise to 32℃, 42℃ and 60℃. Among them, 60℃ is enough to produce irreversible killing effects on cancer cells (if the temperature exceeds 43℃, tumor cells will be pathological, intracellular proteins will be misfolded, and long-term exposure to this temperature will cause irreversible damage to tumor cells. If the temperature exceeds 60℃, the proteins in the tumor cells will coagulate and the tumor cells will be thermally ablated).

[0061] Example 7: Polyethylene glycol-modified selenocyanine compound CySe-mPEG 5K The cell phototherapy effect of CySe-mPEG was investigated by referring to the MTT experimental method of Example 5. 5K The results are as follows Figure 6 As shown in (a) and (b). In the dark, even 64 μM CySe-mPEG 5K After incubation with cells for 24 hours, the cell survival rate was still as high as 87%, proving that CySe-mPEG 5K The dark toxicity of the small molecule CyX-NEt2 series dyes is relatively large. 50 The values ​​were 12.08μM, 8.02μM and 5.33μM respectively, which will inevitably cause dark toxicity damage to normal tissues in living organisms. It is worth noting that when using 500mW / cm 2 After 5 min of 750 nm laser irradiation, CySe-mPEG 5K The survival rate of cells incubated with 50 As low as 0.85 μM, indicating that compared with the small molecule CyX-NEt2 series dyes, CySe-mPEG 5K The photostability, biosafety and photoefficacy of the proposed method should be more suitable for in vivo tumor treatment.

[0062] Example 8: Polyethylene glycol-modified selenocyanine compound CySe-mPEG 5KTumor-targeted fluorescence / photoacoustic imaging experimental protocol: 6-8 week-old female mice bearing 4T1 cell tumors were used for fluorescence in vivo imaging, anesthetized with isoflurane gas, and in vivo imaging was performed using a PerkinElmer IVIS Lumina K Series III imaging system. The excitation light for the imaging experiment was 780 nm, and the emission light of the 845 nm channel was collected.

[0063] In order to study CySe-mPEG 5K Can it be used for in vivo tumor imaging? The control small molecule CySe-NEt2 (100 μM, 100 μL) and CySe-mPEG were injected into the tail vein. 5K (100 μM, 100 μL) was injected into BALB / c mice bearing 4T1 tumors and then fluorescence and photoacoustic signals were collected. Figure 7 The results of the in vivo fluorescence imaging capability study are shown in Figure 7 As shown in (a), it can be seen that there was no fluorescence in the tumor site of the mouse before injection. After administration, the fluorescence signal of the control group was mainly concentrated in the liver area and was not enriched in the tumor site. 5K The fluorescent signal can be quickly detected in the tumor site of the mice in the group, which indicates that CySe-mPEG 5K Nanoparticles can be enriched in the tumor area through the tumor EPR effect. The fluorescence signal in the tumor area reaches the maximum value at about 0.5 hours and then decreases ( Figure 7 (a), (c)). After 1 hour, 2 hours and 3 hours of drug administration, the mice in the experimental group were dissected to obtain tumor tissues and other normal tissues for fluorescence photography. The results showed that the fluorescence signal was strongest in the tumor site, and fluorescence signals could also be observed in the liver, spleen, lungs and kidneys. The fluorescence signals were stronger in the liver and kidneys, which may be related to the CySe-mPEG 5K Metabolic pathways related to Figure 7 (b)). Experiments show that CySe-mPEG 5K It can target tumor tissue and its fluorescence can penetrate living tissue for tumor tissue fluorescence imaging.

[0064] Determination of CySe-mPEG in fluorescence imaging mode 5K After having good tumor targeting and imaging capabilities, CySe-mPEG 5K The imaging ability of the photoacoustic imaging mode was investigated using a photoacoustic / ultrasound (PA / US Vevo LAZR-XFujifilm VisualSonics) in vivo imaging system. Similar to fluorescence imaging, the photoacoustic signal PA780 of the tumor area under a 780nm laser was first collected, such as Figure 7As shown in (f) and (g), compared with the photoacoustic signal of the tumor area at 0 hours, the photoacoustic signal PA780 in the tumor area became significantly stronger after 1 hour of administration and could be observed for several hours. However, the photoacoustic signal of the tumor of the mice injected with the small molecule CySe-NEt2 control group did not change significantly. The above results are consistent with fluorescence imaging. This shows that CySe-mPEG 5K It can effectively enrich in the tumor area, realize the imaging of the tumor area under the photoacoustic mode, and provide more accurate spatiotemporal information for clinical treatment of tumors.

[0065] Example 9: Polyethylene glycol-modified selenocyanine compound CySe-mPEG 5K Experimental protocol for photothermal imaging of tumors:

[0066] Female mice bearing 4T1 tumors aged 6-8 weeks were used for in vivo photothermal imaging using an American FLIE infrared thermal imager. 5K (100 μM, 100 μL, group 2) was injected into tumor-bearing mice and then 0.5 W / cm 2 The 780nm laser was used to illuminate the tumor area and the temperature of the tumor area was recorded.

[0067] In view of the excellent photothermal effect of the material solution and the CySe-mPEG 5K It has good tumor enrichment ability, and it is speculated that it can be used for photothermal therapy of tumor tissue. Near-infrared light has good penetration in biological tissues and can kill deeper tumor tissues. The in vivo photothermal effect of the material was evaluated on 4T1 mice. Mice were injected with PBS, CySe-NEt2 and CySe-mPEG 5K After 1 hour, the 2 ) Laser irradiates the tumor site, and a thermal imager is used to track and record the temperature of the tumor site. Figure 8 As shown, injection of CySe-mPEG 5K The temperature of the mouse tumor site rose rapidly to about 53°C within two minutes and maintained this temperature. At this temperature, the tumor cells were irreversibly killed. In contrast, the temperature of the tumor after injection of PBS solution and CySe-NEt2 and illumination only rose to 34°C and 41°C, and the temperature did not exceed the tumor cell damage threshold (43°C), and did not cause damage to the tumor area. The experimental results show that CySe-mPEG 5K The material can be used for photothermal therapy of mouse tumors.

[0068] Example 10: Polyethylene glycol-modified selenocyanine compound CySe-mPEG 5KEvaluation of tumor phototherapy

[0069] Experimental plan: 6-8 week-old mice bearing 4T1 cell tumors were used to evaluate the effect of photodynamic therapy. The tumor-bearing mice were randomly divided into 3 experimental groups. PBS solution (group 1), CySe-NEt2 (200μM, 100μL, group 2) and CySe-mPEG were injected into the tail vein. 5K (200 μM, 100 μL, group 3) were treated in the dark or with 0.5 W / cm 2 The tumor area was illuminated with a 750nm laser for 10 minutes; the mice were treated every two days, and the drug administration and illumination were stopped after two weeks, and the tumor volume and mouse weight were recorded. The tumor volume was measured with a vernier caliper, tumor volume = tumor length × tumor width 2 × 0.5, and the mouse weight was measured.

[0070] H&E staining experiment of tumor and organ tissue sections: H&E staining is also called hematoxylin-eosin staining. Hematoxylin is alkaline and can dye nucleic acids in cell nuclei into purple-blue; eosin dye is acidic and mainly dyes cell lines and extracellular matrix into red. After treating tumor-bearing mice, the mice were killed according to the regulations, and the tumors, heart, liver, spleen, lungs, kidneys and other major organs of each group of mice were taken and immersed in formalin. After paraffin sections, H&E staining was performed to study the pathological conditions of tumors and major organs.

[0071] The experimental results are as follows Fig. 9 In (b), the tumor volume of mice in control group 1 and control group 2 increased, and their growth rates were similar, indicating that 750nm light irradiation and injection of PBS solution / CySe-NEt2 under light-protected conditions had little effect on the development of mouse tumors. 5K After irradiation with 750nm laser, the tumors in the mice disappeared completely, leaving a black scab at the tumor site. After the scab fell off, new skin grew ( Fig. 9 (c)). Fig. 9 In (a) and (d), the tumor volume and mouse body weight data showed that CySe-mPEG 5K It has excellent photothermal treatment ability for tumors, good biocompatibility and low biological toxicity. There is no significant weight loss in mice during the treatment process.

[0072] CySe-mPEG 5K As a potential material for photothermal therapy of tumors, it is very necessary to study its biological toxicity. 5K Whether it will cause inflammation or damage to other organs. The hearts, livers, spleens, lungs, and kidneys of mice in the control and treatment groups were taken for H&E staining. Fig. 9In (e), the liver cells of mice injected with CySe-NEt2 (group 1) showed inflammation and cavitation in the light-protected and light-exposed groups, which may be due to the high dark toxicity of CySe-NEt2, which is enriched and stagnated in the liver. The myocardial cell structure of mice in other control and treatment groups was normal, without obvious edema. There was no obvious inflammation in the liver cells, the alveoli and bronchial structures were clear, the alveolar wall blood vessels were not congested, there was no obvious fibrosis and inflammation symptoms, and no obvious damage was observed in other tissues. At the same time, only the injection of CySe-mPEG 5K The tumors of mice in the light-irradiated group showed cell necrosis and apoptosis. 5K It has good biocompatibility and low toxicity, has no obvious toxicity to mice at the dose used in the experiment, and can be used for photothermal therapy of living tumors.

Claims

1. A selenocyanine compound, characterized in that It has the following structure: , wherein R represents N,N-diethylamino; X - Represents anion.

2. The selenocyanine compound according to claim 1, characterized in that The X - Selected from Cl - Br - ,I - 、NO3 - or PF4 - .

3. A polyethylene glycol-modified selenocyanine compound, characterized in that It has the following structure: , wherein R represents N,N-diethylamino; X - Represents anion, n=an integer from 3 to 200.

4. The polyethylene glycol-modified selenocyanine compound according to claim 3, characterized in that R represents N,N-diethylamino, n=79.

5. The polyethylene glycol-modified selenocyanine compound according to claim 4, characterized in that The X - Selected from Cl - Br - ,I - 、NO3 - or PF4 - .

6. Use of the selenocyanine compound according to claim 1 or 2 or the polyethylene glycol-modified selenocyanine compound according to claim 3 or 4 in the preparation of fluorescent dyes or tumor phototherapy drugs.

7. The use according to claim 6, characterized in that The fluorescent dye is a fluorescent probe for cell and living body imaging.

Images