Organic small-molecule optical diagnosis and treatment reagent, nanoparticle and preparation method and application thereof

Abstract

The application discloses an organic small-molecule optical diagnosis and treatment reagent, nanoparticles and a preparation method and application thereof. The organic small-molecule optical diagnosis and treatment reagent has high near-infrared second region fluorescence brightness, good photo-thermal conversion effect and can generate type I active oxygen. The nanoparticles have good water solubility and biocompatibility, excellent near-infrared second region fluorescence imaging capability, type I photodynamic and photo-thermal conversion performance. Under single near-infrared laser excitation, the nanoparticles can be used for preparing a type I photodynamic / photothermal combined treatment reagent under the guidance of near-infrared second region fluorescence imaging, and have good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bio-optical diagnosis and treatment, and more particularly relates to an organic small-molecule optical diagnosis and treatment reagent, a nanoparticle, and a preparation method and application thereof. BACKGROUND

[0002] Cancer is one of the major diseases that seriously threaten human health in the world today. Although modern medical technology is developing rapidly, it is still challenging to completely cure cancer. Therefore, people have been working hard to find new imaging methods and cancer treatment approaches. In recent years, the light diagnosis and treatment system has attracted widespread attention from researchers because it can realize both functions by organically integrating optical imaging technology and optical treatment technology. Fluorescence imaging has attracted widespread attention due to its fast response, high resolution, and high sensitivity. However, traditional fluorescence imaging mainly focuses on the near-infrared region (700-900 nm), which has defects such as low signal-to-noise ratio, insufficient spatial resolution, and poor penetration depth. Near-infrared region II (1000-1700 nm) fluorescence has greatly reduced light scattering, light absorption, and biological autofluorescence in biological tissues, thus having better tissue penetration depth and higher imaging signal-to-noise ratio, and having good application prospects in the fields of tumor diagnosis and angiography.

[0003] Optical therapy is a kind of light-mediated non-invasive and harmless cancer treatment, mainly including photodynamic therapy and photothermal therapy. Compared with traditional therapy, the optical therapy process can selectively act on the tumor area, reduce drug toxicity, and improve treatment effect. Photodynamic therapy usually uses photosensitizers to generate highly cytotoxic reactive oxygen species under the irradiation of a certain excitation light source to kill cancer cells; photothermal therapy uses light-heat agents to convert external light into heat to ablate cancer cells. At present, a series of organic small-molecule near-infrared region II optical diagnosis and treatment probes have been designed and developed, but the reactive oxygen species generated by these probes are mostly type II (i.e. singlet oxygen, which depends on oxygen for generation), and the hypoxic microenvironment of tumors greatly limits the generation of singlet oxygen, which is not conducive to efficient tumor treatment. In contrast, the generation of type I reactive oxygen species (hydroxyl radicals, superoxide anions, etc.) has lower dependence on oxygen, so type I reactive oxygen species can greatly overcome the hypoxic microenvironment of tumors and have better tumor treatment effect. In addition, the reported probes also have defects such as low near-infrared region II fluorescence brightness and poor photothermal effect, so it is necessary to design and prepare a high-performance organic small-molecule optical diagnosis and treatment agent that has both near-infrared region II fluorescence imaging and type I photodynamic / photothermal performance. SUMMARY

[0004] The present application aims at the above-mentioned deficiencies, and provides an organic small molecule optical diagnosis and treatment reagent, a nanoparticle and a preparation method and application thereof, the nanoparticle can effectively generate near-infrared two-region fluorescence, type I active oxygen and photothermal under 808 nm laser irradiation, thereby being used for preparing a type I photodynamic / photothermal combined treatment reagent guided by near-infrared two-region fluorescence imaging.

[0005] To achieve the above-mentioned purposes, the present application is realized by the following technical solutions:

[0006] An organic small molecule optical diagnosis and treatment reagent has the following structural formula:

[0007]

[0008] The synthesis route of the above-mentioned organic small molecule optical diagnosis and treatment reagent is as follows:

[0009]

[0010] The preparation method of the above-mentioned organic small molecule optical diagnosis and treatment reagent comprises the following steps:

[0011] Compound 2, compound 3 and a palladium catalyst are dissolved in an organic solvent, and reacted at 100-120 DEG C under nitrogen protection for 12-15 h, and compound 4 is obtained after purification;

[0012] n-BuLi is added to an organic solution of compound 5, stirred at-75 DEG C to-80 DEG C for 1.2-2.0 h, then compound 4 is slowly added to the organic solution, stirred after being warmed to room temperature, reacted for 12-15 h, the organic solvent is spun off, octane, acetic acid and concentrated sulfuric acid are added and stirred for 4-5 h, and compound 6 is obtained after extraction, drying and purification;

[0013] n-BuLi is slowly added to an organic solution of compound 6 at-75 DEG C to-80 DEG C, stirred under nitrogen protection for 1.2-2.0 h, then tributyltin chloride is slowly added, stirred after being warmed to room temperature, reacted for 3-5 h, and compound 7 is obtained after extraction and spinning off the organic solvent;

[0014] Compound 7, compound 8 and a palladium catalyst are dissolved in an organic solvent, and reacted at 100-120 DEG C under nitrogen protection for 17-20 h, and compound 9 is obtained after purification;

[0015] Compound 9 and compound 10 are dissolved in an organic solvent, triethylamine is slowly added dropwise, and reacted at 60-65 DEG C for 12-15 h, and the organic small molecule optical diagnosis and treatment reagent of formula (1) is prepared after chromatographic purification after the reaction is completed.

[0016] Preferably, in the above preparation method, the molar ratio of the compound 2 and the compound 3 is (1:2.2)-(1:2.6); the molar ratio of the compound 4 and the compound 5 is (1:6.0)-(1:7.0); the molar ratio of the compound 6 and tributyltin chloride is (1:2.0)-(1:2.5), the molar ratio of the compound 7 and the compound 8 is (1:2.5)-(1:3.5), and the molar ratio of the compound 9 and the compound 10 is (1:4.0)-(1:5.5).

[0017] The application further provides a nanoparticle of an organic small-molecule optical diagnosis and treatment reagent, which is prepared by using an amphiphilic polymer F127 and a nanoscale coprecipitation method based on the organic small-molecule optical diagnosis and treatment reagent.

[0018] The nanoparticle of the organic small-molecule optical diagnosis and treatment reagent has a maximum light absorption wavelength of 790 nm in an aqueous solution, and has a fluorescence emission peak at 934 nm and 1008 nm after excitation by an 808 nm light source, so that the nanoparticle can be used for near-infrared two-region fluorescence imaging; under excitation by the 808 nm light source, the nanoparticle has type I active oxygen generation and photothermal effects, and can be used for preparation of a tumor type I photodynamic / photothermal combined treatment reagent under guidance of near-infrared two-region fluorescence imaging.

[0019] The preparation method of the nanoparticle of the organic small-molecule optical diagnosis and treatment reagent comprises the following steps:

[0020] The organic small-molecule optical diagnosis and treatment reagent is dissolved in THF to obtain a solution, the solution is added to a deionized water solution of F127, an ultrasonic cell crusher is used for oscillation for 5 min, after ultrasonic oscillation, the solution is stirred in a fume hood for 12 h to completely volatilize THF, and then impurities are filtered by using a water-based filter membrane to obtain a nanoparticle solution of the organic small-molecule optical diagnosis and treatment reagent.

[0021] The application further provides application of the organic small-molecule optical diagnosis and treatment reagent in preparation of a NIR-II fluorescence imaging contrast agent under laser irradiation, that is, the nanoparticle aqueous solution is used to prepare the NIR-II fluorescence imaging contrast agent.

[0022] The application further provides application of the organic small-molecule optical diagnosis and treatment reagent in preparation of a tumor type I photodynamic / photothermal combined treatment reagent triggered by 808 nm laser, that is, the nanoparticle is used to prepare the tumor type I photodynamic / photothermal combined treatment reagent triggered by 808 nm laser.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The organic small-molecule optical diagnosis and treatment reagent has high near-infrared two-region fluorescence brightness, good photo-thermal conversion effect, and can generate type I reactive oxygen, and has a wide application prospect as an imaging probe and a high-efficiency tumor treatment reagent.

[0025] The nanoparticle preparation method of the organic small-molecule optical diagnosis and treatment reagent is simple in operation, good in effect, and has good biocompatibility and water solubility. The nanoparticle has excellent near-infrared two-region fluorescence imaging capability, type I photodynamic and photo-thermal conversion performance, and can be used for preparation of a type I photodynamic / photothermal combined treatment reagent under the guidance of near-infrared two-region fluorescence imaging, and has a good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A mass spectrum of the organic small-molecule optical diagnosis and treatment reagent prepared in Example 1;

[0027] Figure 2 A UV spectrum of an aqueous solution of the organic small-molecule optical diagnosis and treatment reagent nanoparticle prepared in Example 2;

[0028] Figure 3 A fluorescence spectrum of the aqueous solution of the organic small-molecule optical diagnosis and treatment reagent nanoparticle prepared in Example 2;

[0029] Figure 4 An in-vitro near-infrared two-region fluorescence imaging diagram of the aqueous solution of the organic small-molecule optical diagnosis and treatment reagent nanoparticle prepared in Example 2;

[0030] Figure 5 A whole-body blood vessel imaging diagram of a mouse after tail vein injection of the aqueous solution of the organic small-molecule optical diagnosis and treatment reagent nanoparticle prepared in Example 2;

[0031] Figure 6 A diagram of change of UV absorption intensity of a mixture of the organic small-molecule optical diagnosis and treatment reagent nanoparticle and DPBF at 414 nm under 1W / m2 light irradiation with light irradiation time;

[0032] Figure 7 A diagram of photo-thermal stability test of the organic small-molecule optical diagnosis and treatment reagent nanoparticle prepared in Example 2. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present application will be described in more detail below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1

[0035] The present embodiment provides an organic small-molecule optical diagnosis and treatment reagent, which has the following structural formula:

[0036]

[0037] The synthesis route of the above-mentioned small organic molecule optical diagnosis and treatment reagent is as follows:

[0038]

[0039] The preparation method thereof specifically comprises the following steps:

[0040] Compound 2 (1 g, 2.63 mmol), compound 3 (2.26 g, 6.05 mmol), and tetrakis(triphenylphosphine)palladium (30 mg, 26.31 μmol) were dissolved in anhydrous toluene (20 mL), and the reaction was carried out at 110°C under nitrogen protection for 13 h. After the reaction was completed, extraction was performed with ethyl acetate (3 x 100 mL), and washing was performed with water three times. The combined organic phase was dried over anhydrous MgSO4. After the solvent was removed, purification was performed using a silica gel column chromatography to obtain white solid compound 4 (0.86 g, 85%);

[0041] n-BuLi (3.10 mL, 7.76 mmol, 2.5M hexane solution) was added to a solution of compound 5 (2.14 g, 7.76 mol) in anhydrous THF (15 mL) at -78°C, and the reaction mixture was stirred at -78°C for 1.5 h. Then, a solution of compound 4 (0.50 g, 1.29 mmol) in THF (20 mL) was slowly added. After being warmed to room temperature and stirred for 13 h, the reaction mixture was poured into water and extracted with ethyl acetate twice. The combined organic phase was dried over anhydrous Na2SO4. The solvent was evaporated, and the yellow residue was dissolved in octane (50 mL) and acetic acid (3 mL). Then, concentrated H2SO4 (0.2 mL) was slowly added dropwise. After being stirred for 4 h, the organic layer was extracted with ethyl acetate (3 x 100 mL) and washed with water three times. The combined organic phase was dried over anhydrous MgSO4. After the solvent was removed, purification was performed using a silica gel column chromatography. Compound 6 (0.96 g, 71%) was obtained in the form of a yellow solid;

[0042] n-BuLi (0.38 mL, 958.4 μmol, 2.5M hexane solution) was slowly added to a solution of compound 6 (0.50 g, 0.48 mmol) in anhydrous THF (20 mL) at -75°C to -80°C. After being stirred for 1.5 h under nitrogen protection, tributyltin chloride (191 mg, 958.4 μmol) was slowly added. After being warmed to room temperature and stirred for 3 h, water was added to quench the reaction, and the reaction mixture was extracted with diethyl ether twice. The organic layer was dried over anhydrous MgSO4. After the solvent was removed, compound 7 was obtained without further purification and was directly used;

[0043] Compound 7 (800 mg, 584 μmol), compound 8 (560 mg, 1.75 mmol) and tetrakis(triphenylphosphine)palladium (7 mg, 5.85 μmol) were dissolved in anhydrous toluene (20 mL) and reacted at 110°C under nitrogen protection for 18 h. After the reaction was completed, purification was performed by silica gel column chromatography to obtain compound 9 (0.52 g, 59%) in the form of an orange solid;

[0044] Compound 9 (150.0 mg, 98.68 μmol) and compound 10 (95.51 mg, 0.49 mmol) were dissolved in CHCl3(8 mL), then triethylamine (0.3 mL) was slowly added dropwise, and the mixture was reacted at 62°C for 13 h. After extraction with dichloromethane, the resulting mixture was washed with water and dried over anhydrous MgSO4. After removal of the solvent, the product, an organic small-molecule optical diagnosis and treatment agent as described in formula (1), was obtained in the form of a black solid (130 mg, 70%) by purification by silica gel column chromatography. The mass spectrum thereof is shown in Figure 1

[0045] Example 2

[0046] The present example provides a method for preparing nanoparticles of the organic small-molecule optical diagnosis and treatment agent described in Example 1, comprising the following steps:

[0047] An organic small-molecule optical diagnosis and treatment agent (1 mg) described in Example 1 was weighed into THF (2.5 mL), and then the solution was quickly added to deionized water (10 mL) in which a amphiphilic substance F127 (20 mg) was dissolved, and the mixture was shaken in an ultrasonic cell crusher for 5 min. After ultrasonic treatment, the solution was blown with nitrogen for 2 h under magnetic stirring to remove THF. After removal of THF, the solution was filtered using a water-based filter membrane to remove impurities and then centrifuged at a speed of 3000 r / min for 20 min. The solution at the bottom of the ultrafiltration centrifuge tube was poured out, and deionized water was added to the sample slot on the centrifuge tube for washing. After the steps of centrifugation were repeated three times, a quantitative aqueous solution of organic small-molecule optical diagnosis and treatment agent nanoparticles was obtained.

[0048] The ultraviolet absorption of the aqueous nanoparticle solution prepared by the above method is shown in Figure 2 , and the maximum absorption peak thereof in the aqueous solution is at 790 nm. The fluorescence emission spectrum of the aqueous nanoparticle solution is shown in Figure 3 , and the fluorescence emission peaks thereof are at 934 nm and 1008 nm.

[0049] The performance test of the aqueous nanoparticle solution prepared by the above method is as follows:

[0050] 1. In vitro near-infrared two-region fluorescence imaging of the aqueous nanoparticle solution of the organic small-molecule optical diagnosis and treatment agent:

[0051] ​The above nanoparticle aqueous solution with a concentration of 0.1 mg / ml was prepared, placed in a small centrifuge tube, and excited by a laser with a wavelength of 808 nm under a near-infrared two-region fluorescence imager to obtain a near-infrared two-region fluorescence image, as shown in Figure 4 The results show that the nanoparticle has excellent near-infrared two-region fluorescence imaging capability.

[0052] 2. In vivo near-infrared two-region fluorescence imaging of the nanoparticle aqueous solution of the organic small-molecule optical diagnosis and treatment agent:

[0053] Figure 5 The nanoparticle aqueous solution of the present application was injected into the tail vein of a mouse for whole-body vascular imaging. The injection dose of the nanoparticle aqueous solution was 100 μL, 1 mg / mL, and the laser was 808 nm. The whole-body blood vessels of the mouse were clearly visible 10 min after injection, indicating that the nanoparticle has excellent in vivo near-infrared two-region fluorescence imaging performance. Subsequently, the mouse was imaged at different time points using a near-infrared two-region fluorescence imager. As time increased, the fluorescence intensity at the tumor site of the mouse increased, indicating that the nanoparticle has good tumor imaging capability.

[0054] 3. Photodynamic performance test of the nanoparticle aqueous solution of the organic small-molecule optical diagnosis and treatment agent

[0055] The nanoparticle aqueous solution was added to a cuvette, and 1,3-diphenyl isobenzofuran (DPBF) ethanol solution was added dropwise. The cuvette was irradiated with a laser (808 nm) with a light power density of 1 W / m 2 , 0.75 W / cm 2 , 0.5 W / cm 2 , and 0.25 W / cm 2 , respectively. The change in the ultraviolet absorption peak of DPBF at 414 nm was recorded, Figure 6 The change trend of the ultraviolet absorption peak of DPBF at 414 nm under 1 W / m 2 light irradiation over time. The absorption peak of DPBF at 414 nm decreased significantly with continuous laser irradiation, thereby reflecting the generation of reactive oxygen species during the irradiation process. The greater the light power density, the greater the decrease in the absorption peak of DPBF at 414 nm, indicating that the nanoparticle has good photodynamic performance. In addition, we used a specific reactive oxygen species probe to identify the types of reactive oxygen species, and the results showed that the nanoparticle can generate hydroxyl radicals and superoxide anions, i.e., the nanoparticle has type I photodynamic effect, which can effectively overcome the limitation of the tumor hypoxic microenvironment and improve the tumor treatment effect.

[0056] 4. Photothermal performance test of the nanoparticle aqueous solution of the organic small-molecule optical diagnosis and treatment agent:

[0057] The photo-thermal performance of the nanoparticle aqueous solution under different solution concentrations and different light power densities is studied.

[0058] The nanoparticle aqueous solutions with concentration gradients of 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml and 100 μg / ml are respectively configured, and the solutions are irradiated by a laser (808 nm, 1 W / cm 2 ) to record the temperature change trend of the solutions with time by an infrared camera. 2 The results show that under the same laser power irradiation, the temperature of the nanoparticle aqueous solution continuously increases with the increase of the concentration. In particular, when the nanoparticle concentration is 100 μg / ml and the laser power is 1 W / cm 2 , the highest temperature can be raised to above 80℃, which indicates the excellent photo-thermal performance of the nanoparticles.

[0059] Then, the nanoparticle solutions with the same concentration are irradiated by a laser (808 nm) with power densities of 0.25 W / cm 2 , 0.5 W / cm 2 , 0.75 W / cm 2 and 1 W / cm 2 , respectively, and the temperature change curves of the solutions with time are detected. Figure 7 The results show that with the increase of the laser power density, the increase of the temperature of the nanoparticle solution is more obvious. In addition, the photo-thermal stability of the nanoparticles is investigated, and as shown in FIG. 4, the highest temperature of the nanoparticles does not change significantly in 4 cycles of laser "on-off", which indicates that the nanoparticles have excellent photo-thermal stability.

[0060] The above has described the embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and technical principles of the described embodiments, and these modifications and changes should also be considered as the protection scope of the present application.

Claims

1. An organic small-molecule optical diagnosis and treatment reagent, characterized in that, The structural formula is as follows: （1）。 2. The method for preparing the organic small-molecule optical diagnosis and treatment reagent according to claim 1, characterized in that, The method comprises the following steps: Compound 2, compound 3 and a palladium catalyst are dissolved in an organic solvent, and reacted at 100-120 DEG C under nitrogen protection for 12-15 hours, to obtain compound 4 after purification; n -BuLi is added to the organic solution of compound 5, stirred at -75°C~ -80°C for 1.2~2.0h, then the organic solution of compound 4 is added, stirred after warming to room temperature, the reaction is carried out for 12~15h, the organic solvent is spun off, octane, acetic acid, concentrated sulfuric acid are added, stirred for 4~5h, after extraction, drying, purification, compound 6 is obtained;​ At -75℃ to -80℃, n -BuLi was added to the organic solution of compound 6, and the reaction was stirred for 1.2 to 2.0 h under nitrogen protection. Then, tributyltin chloride was added, and the mixture was heated to room temperature and stirred for 3 to 5 h. After extraction and removal of organic solvent, compound 7 was obtained. Compound 7, compound 8 and a palladium catalyst are dissolved in an organic solvent, and reacted at 100-120 DEG C under nitrogen protection for 17-20 hours, to obtain compound 9 after purification; Compound 9 and compound 10 are dissolved in an organic solvent, triethylamine is added dropwise, and the reaction is carried out at 60-65 DEG C for 12-15 hours; after the reaction is completed, chromatographic purification is carried out, to obtain the organic small-molecule dye of formula (1); The structural formula of the above compound 2 is: ; The structural formula of compound 3 is: ; The structural formula of compound 4 is: ; The structural formula of compound 5 is: ; The structural formula of compound 6 is: ; The structural formula of compound 7 is: ; The structural formula of compound 8 is: ; The structural formula of compound 9 is: ; The structural formula of compound 10 is: .

3. The method for preparing the organic small molecule optical diagnostic reagent according to claim 2, characterized in that, The molar ratio of compound 2 to compound 3 is (1:2.2)-(1:2.6); the molar ratio of compound 4 to compound 5 is (1:6.0)-(1:7.0); the molar ratio of compound 6 to tributyltin chloride is (1:2.0)-(1:2.5); the molar ratio of compound 7 to compound 8 is (1:2.5)-(1:3.5); and the molar ratio of compound 9 to compound 10 is (1:4.0)-(1:5.5).

4. A nanoparticle of an organic small-molecule optical diagnostic and therapeutic agent, characterized in that, The organic small-molecule optical diagnosis and treatment reagent is based on the organic small-molecule optical diagnosis and treatment reagent of claim 1, and is prepared by using an amphiphilic polymer F127 and a nano-coprecipitation method.

5. A method for preparing the nanoparticles of the organic small-molecule optical diagnostic and therapeutic agent according to claim 4, characterized by, The method comprises the following steps: The organic small-molecule optical diagnosis and treatment reagent of claim 1 is dissolved in THF, and the prepared solution is added to a deionized water solution of F127; after ultrasonic oscillation, THF is removed to obtain a nano-particle solution of the organic small-molecule optical diagnosis and treatment reagent.

6. Use of the organic small-molecule optical diagnosis and treatment reagent of claim 1 in preparation of a NIR-II fluorescence imaging contrast agent under laser irradiation.

7. Use of the organic small-molecule optical diagnosis and treatment reagent of claim 1 in preparation of a tumor type I photodynamic / photothermal combined treatment reagent triggered by 808 nm laser.

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