A near-infrared heptamethine cyanine dye and its application in the field of photothermal therapy

Abstract

The application discloses a near-infrared heptamethine cyanine dye and application thereof in the field of photothermal therapy. 2000 After being wrapped into nanoparticles, the photothermal stability of the heptamethine cyanine dye is greatly enhanced, no photobleaching occurs in four photothermal cycle experiments, the heptamethine cyanine dye has high efficient photothermal conversion capacity, and the photothermal conversion efficiency is as high as 82%. The photothermal reagent prepared from the heptamethine cyanine dye has significant cell killing capacity at low concentration and low light dose under light excitation, and has low dark toxicity. The near-infrared heptamethine cyanine dye provided by the application has simple structure, simple synthesis mode and excellent clinical application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of materials and biomedicine, and particularly relates to heptamethine cyanine dye nanoparticles and application as photothermal agents in photothermal therapy. BACKGROUND

[0002] Cancer is one of the highest morbidity and mortality diseases in the world. According to statistics, nearly 100 million cases (or nearly one sixth) of death in 2020 were caused by cancer. The most common cancers are breast cancer, lung cancer, colon cancer, rectal cancer and prostate cancer. In view of the high risk and mortality, researchers all over the world have been working hard to develop more accurate, rapid diagnostic strategies and effective treatment methods to combat cancer. Traditional treatment methods bring great toxic side effects to patients and cannot completely remove the lesion site, with the risk of recurrence. The ultimate goal of cancer is still focused on developing non-invasive treatment methods that can effectively detect cancer and cell metastasis and timely treatment at an early stage

[0003] PTT is considered an advanced treatment method that can provide non-invasive, accurate and rapid disease diagnosis and treatment. By adjusting the light dose of the external excitation light source and the concentration of the photothermal agent (PTA), the toxic side effects are reduced and the damage to the surrounding healthy tissues is minimized. The treatment method of PTT is achieved by using PTAs. The corresponding wavelength of excitation light is used to irradiate PTAs, and the absorbed light energy is converted into heat. The local tissue temperature rises, exceeding the temperature threshold (42℃) that tumor cells can withstand, killing tumor cells and ablating tumors. During the treatment process, the photothermal effect promotes the expansion of the surrounding tissues, generates ultrasonic waves, and is converted into photoacoustic image (PAI) signals. PAI has high resolution and tissue penetration depth, which can realize real-time imaging of the lesion site and achieve accurate photothermal treatment.

[0004] Heptamethine cyanine dye has a high molar extinction coefficient and absorbs near-infrared light with a wavelength of 700 nm or more, which can penetrate deeper tissues to reach the lesion site. However, so far, most of the reported cyanine dye photothermal agents have low photothermal conversion efficiency and are prone to photobleaching after excitation light irradiation, which cannot exist stably. In order to enhance the photothermal conversion efficiency and stability of cyanine dye, 2-mercaptobenzothiazole is introduced as a rotor unit in the middle to enhance non-radiative transition in the form of intramolecular motion, thereby improving the photothermal conversion efficiency. In addition, DSPE-PEG 2000 The nanoparticles are prepared by nanocoprecipitation method to further enhance the biocompatibility and photothermal stability. The nanoparticles have good application potential in the field of photothermal therapy. SUMMARY

[0005] The application discloses a heptamethine cyanine dye, and provides a heptamethine cyanine dye nanoparticle photothermal reagent.

[0006] The technical scheme of the application is a compound of formula I

[0007]

[0008] X and Y are independently N, O or S.

[0009] Z - is an anion selected from F - , Cl - , Br - , I - , PF6 - , NO3 - , SO4 2- , CF3SO3 - . Z - has the same negative charge as the positive charge of the cation in formula I.

[0010] A specific compound, X and Y are independently N, O or S, and X and Y are not the same.

[0011] A nanoparticle comprising at least one of the above-mentioned compounds of formula I.

[0012] A specific nanoparticle, wherein the compound is loaded in a nanoparticle composed of DSPE-PEG 2000 .

[0013] In a specific technical scheme, the diameter of the nanoparticle is 50-150 nm.

[0014] A photothermal reagent comprising at least one of the above-mentioned compounds of formula I.

[0015] The preparation method of the photothermal reagent, wherein a solution of the compound is added dropwise into a DSPE-PEG 2000 (di-stearoyl phosphatidyl ethanolamine-polyethylene glycol) aqueous solution under ultrasonic state, and ultrasonic treatment is performed for 20-60 min; a dialysis bag is used to dialyze small molecule compounds which are not encapsulated into nanoparticles, so that the photothermal reagent is obtained.

[0016] In a specific preparation method, the compound can be dissolved in an organic solvent such as DMF or DMSO, and DSPE-PEG 2000 is dissolved in deionized water.

[0017] A specific preparation method, the compound and DSPE-PEG 2000 The mass ratio is 0.1-0.2:1.

[0018] A specific preparation method of the photothermal agent, the nanoparticles are dispersed in water, PBS or common medical solvents.

[0019] A specific compound, the structure is as follows:

[0020]

[0021] The application also provides a specific preparation method of heptamethine cyanine dye, and the steps are as follows:

[0022]

[0023] (1) Synthesis of compound 1

[0024] After stirring anhydrous DCM and anhydrous DMF in an ice water bath for 30 min, anhydrous DCM containing POCl3 is slowly added dropwise into the above-mentioned mixed system, the dropwise adding time is controlled within 30 min, cyclohexanone is added into the reaction system under a nitrogen atmosphere, and the temperature is increased to 80 DEG C and reacted for 5 h. After the reaction is completed, the solution is added dropwise into ice water to precipitate a light yellow precipitate, which is filtered, washed with water, and dried under vacuum to obtain compound 1, which is directly used in the next step without purification.

[0025] (2) Synthesis of compound 2

[0026] 2,3,3-trimethyl-3H-indole is dissolved in toluene and stirred under reflux, and the reaction is carried out overnight. After being cooled to room temperature, the reaction solution is slowly added dropwise into ether to precipitate a solid, which is filtered, washed with ether, and dried under vacuum to obtain purple red solid product 2, which is directly used in the next step without purification.

[0027] (3) Synthesis of compound 3

[0028] Compound 1, compound 2 and sodium acetate are dissolved in acetic anhydride, and the reaction is stirred at 60 DEG C under a nitrogen atmosphere for 1 h. After the reaction is completed, ether is added, and the solution is refrigerated in a refrigerator for 2 h, and then filtered under suction to obtain golden yellow solid, which is dried under vacuum and purified by column chromatography to obtain golden yellow compound 3.

[0029] (4) Synthesis of compound 4

[0030] Compound 3, 2-mercaptobenzothiazole and triethylamine are dissolved in DMF, and the reaction is stirred at room temperature under a nitrogen atmosphere overnight. After the reaction is completed, the reaction solution is added dropwise into ether, and the solution is refrigerated in a refrigerator for 2 h, and then filtered under suction, and purified by column chromatography to obtain green solid compound 4.

[0031] A specific preparation method of the photothermal agent:

[0032] Compound 4 is dissolved in an organic solvent, and a water solution containing DSPE-PEG 2000 is added dropwise under ultrasonic state for 20-60 minutes, and a dialysis bag is used for dialysis treatment to obtain a water solution containing nanoparticles.

[0033] The above-mentioned heptamethine cyanine dye, nanoparticles or photothermal reagent can be applied in the field of photothermal therapy.

[0034] Compared with the prior art, the present application has the following outstanding advantages: (1) the synthesis process of the photothermal reagent is simple and easy to operate; (2) the nanoparticles have good water solubility, excellent photothermal stability and photothermal conversion capacity; (3) the nanoparticles prepared by the present application can cause significant killing ability to tumor cells at low concentration and low light dose, and are expected to be applied in in vivo experiments. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of compound 4.

[0036] Figure 2 is the ultraviolet absorption spectrum diagram of the nanoparticles of example 1 of the present application.

[0037] Figure 3 is the TEM diagram of the nanoparticles of example 1 of the present application.

[0038] Figure 4 is the in vitro photothermal heating image of the nanoparticles of example 1 of the present application under laser (808nm, 500mW / cm 2 ) irradiation within 5 minutes.

[0039] Figure 5 is the photothermal cycle experiment of the nanoparticles of example 1 of the present application under laser (808nm, 500mW / cm 2 ) irradiation.

[0040] Figure 6 is the cell phototoxicity experiment result diagram of the nanoparticles of example 1 of the present application. DETAILED DESCRIPTION

[0041] Example 1: Preparation and characterization of photothermal reagent

[0042]

[0043] (1) Synthesis of compound 1

[0044] After stirring anhydrous DCM (21 mL, 163.8 mmol) and anhydrous DMF (21 mL) under nitrogen atmosphere for 30 min in an ice bath, POCl3(21 mL, 138 mmol) dissolved in anhydrous DCM (7.5 mL) was slowly added dropwise to the above mixture, and the dropwise addition was controlled to be completed within 30 min. Cyclohexanone (6 g, 60 mmol) was added to the reaction mixture, and the reaction was performed at 80°C for 5 h. After the reaction was completed, the solution was added dropwise to ice water to precipitate a light yellow solid, which was filtered, washed with water, and dried in vacuo to obtain compound 1 (8.52 g, 84.3%), which was used in the next reaction without purification.

[0045] (2) Synthesis of compound 2

[0046] Compound 2 (6.6 g, 58%) was obtained as a purple red solid by refluxing and stirring 2,3,3-trimethyl-3H-indole (4 g, 25 mmol) and iodoethane (2.4 mL, 30 mmol) in 20 mL of toluene, and then precipitating a solid by slowly adding the reaction solution to diethyl ether, filtering, washing with diethyl ether, and drying in vacuo, which was used in the next reaction without purification.

[0047] (3) Synthesis of compound 3

[0048] Compound 3 (1.85 g, 66.1%) was obtained as a golden yellow solid by stirring compound 1 (0.75 g, 4.4 mmol), compound 2 (2.77 g, 8.8 mmol), and sodium acetate (0.36 g, 4.4 mmol) in 10 mL of acetic anhydride at 60°C for 1 h, and then precipitating a solid by adding 100 mL of diethyl ether and storing in a refrigerator for 2 h, filtering, drying in vacuo, and purifying by column chromatography.

[0049] (4) Synthesis of compound 4

[0050] Compound 4 (40 mg, 31.8%) was obtained as a green solid by stirring compound 3 (124.78 mg, 195.25 mmol), 2-mercaptobenzothiazole (326.55 mg, 1.95 mmol), and 100 μL of triethylamine in 5 mL of DMF at room temperature overnight, adding the reaction solution dropwise to 200 mL of diethyl ether, storing in a refrigerator for 2 h, filtering, and purifying by column chromatography (methanol: dichloromethane = 1:100). 1H NMR(500MHz,DMSO)δ8.64(s,2H),7.91(s,2H),7.57(s,2H),7.42(s,4H),7.33(s,1H),7.25(s,2H),6.39(s,2H),5. 76(s,1H),4.25(s,4H),2.82(s,4H),1.97(s,2H),1.50(s,12H),1.29(s,6H).HRMS:found:642.2971,calculated:C 41 H 44 N3S2 + ,642.2969.

[0051] (5) Weigh 1 mg of compound 4 and dissolve it in DMF (1 mL). Add DSPE-PEG dropwise under sonication. 2000 (10mg) was added to 10ml of Wahaha purified water, sonicated for half an hour, and dialyzed for 3 days using a 3500 molecular weight dialysis bag, with the water changed twice a day, to obtain an aqueous solution of nanoparticles (nanophotothermal reagent). The DSPE-PEG2000 used in this example was purchased from Anaiji.

[0052] Test Example: In this test example, Wahaha purified water was used for dilution.

[0053] The concentration of the nano-photothermal reagent prepared in Example 1 was calibrated:

[0054] A stock solution of compound 4 with a concentration of 5 mmol / L was prepared. 0.15, 0.3, 0.45, 0.6, and 0.9 μL of this stock solution were added to 3 mL of water, respectively. The corresponding absorption wavelengths were measured using a Perkin Elmer Lambda 35UV-Vis spectrometer. A standard concentration-absorption curve for the small molecule was linearly fitted based on the different absorbance values ​​at the same wavelength. Simultaneously, 10 μL, 30 μL, and 50 μL of the nano-aqueous solution prepared in Example 1 were diluted to 3 mL using a pipette, and the UV absorption spectra were measured. Subsequently, quantification was performed based on the standard curve, and the molar concentration of the dye in the photothermal reagent prepared in Example 1 was calculated to be 73 μmol / L.

[0055] The morphology of nanoparticles was imaged using a transmission electron microscope (HT7700, Hitachi, Japan), such as... Figure 3 As shown, the nanophotothermal reagent of Example 1 was successfully encapsulated into nanoparticles.

[0056] In vitro photothermal heating experiment of nano-photothermal reagent:

[0057] The pipette gun pipettes 685 μL of the nano-photothermal reagent prepared in Example 1 into 315 μL of deionized water to prepare a 50 μmol / L concentration of the nanoparticle aqueous solution. An 808 nm laser (Changchun Xin Industry Optoelectronic Technology) is used to irradiate the solution at a light power density of 500 mW / cm 2 The laser irradiates the 50 μmoL / L nano-aqueous solution, and the temperature rise trend within 5 min is determined using an infrared camera (FLIR-1910582), as shown in FIG. 5. The nano-photothermal reagent can be heated to above 72°C within 5 min. Figure 4

[0058] In vitro photothermal stability experiment of the nano-photothermal reagent:

[0059] The 50 μmoL / L concentration of the nanoparticle solution is prepared according to the above method. An 808 nm laser (Changchun Xin Industry Optoelectronic Technology) is used to irradiate the solution at a light power density of 500 mW / cm 2 After 5 min of irradiation, the solution is cooled to the initial temperature at room temperature, and the same conditions are used for excitation and irradiation. This process is repeated four times. As shown in FIG. 6, the nano-photothermal reagent does not undergo photobleaching and has excellent photothermal stability. Figure 5

[0060] In vitro photothermal treatment experiment of the nano-reagent:

[0061] 4T1 cells are selected for the cytotoxicity experiment. The specific operation steps are as follows: 4T1 cells are inoculated into a 96-well plate, and 100 μL of DMEM medium is added to each well for incubation. When the cell density is about 80%, DMEM medium is used as the solvent to prepare nano-photothermal reagent solutions with different concentrations (0 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM). The experiment is divided into 8 groups, 4 wells for each group. 100 μL of the nano-solution with the corresponding concentration is added to each well, and the 4T1 cells are incubated for 3 h. The light irradiation group is irradiated for 5 min using an 808 nm laser (light power is 400 mW / cm 2 ). Figure 6 As can be seen, the light irradiation group of 0.2 μM has killed 50% of the cells, and the IC50 value is 0.2 μM. The cell survival rate is reduced to below 30% at a concentration of 0.7 μM, while the corresponding dark toxicity cell survival rate is above 75%. The nano-photothermal reagent can effectively kill tumor cells at a concentration of 0.7 μmol / mL. The above results show that the nano-photothermal reagent has excellent cell phototoxicity and low dark toxicity, and exhibits excellent cell killing ability at a low concentration and low light power density. It is a very potential photothermal reagent.​​

Claims

1. A photothermal reagent, characterized in that, Compounds containing the following structures: ； Where X and Y are N and S atoms, respectively; Z − The anion is selected from F. − Cl − ,Br − I − PF6 − NO3 − SO4 2− CF3SO3 − One of them; A solution of the compound was added dropwise to DSPE-PEG under ultrasonic conditions. 2000 The photothermal reagent was obtained by sonication in an aqueous solution for 20-60 minutes and then dialyzed using a dialysis bag.

2. The photothermal reagent according to claim 1, characterized in that, The compound and DSPE-PEG 2000 The mass ratio is 0.1 to 0.2:

1.

3. The application of the photothermal reagent as described in claim 1 in the preparation of antitumor drugs.

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