阅读说明：本技术 一种pH可逆激活型光热/光动力/荧光一体化探针分子 (PH reversible activation type photo-thermal/photodynamic/fluorescent integrated probe molecule ) 是由 赵旭 赵凯超 严秀平 于 2020-05-15 设计创作，主要内容包括：本发明公开了一种pH可逆激活型光热/光动力/荧光一体化探针分子,属于生物医药技术领域。本发明以pH响应型不对称菁类结构为核心单元,通过结构修饰及在共轭体系中引入卤素等重原子,构建可被肿瘤弱酸性微环境特异性激活的光热/光动力/荧光一体化探针分子。本发明光热/光动力/荧光一体化探针具有良好的稳定性、光热效果以及优越的产生活性氧的能力,在肿瘤治疗方面具有巨大潜力。(The invention discloses a pH reversible activation type photo-thermal/photodynamic/fluorescent integrated probe molecule, and belongs to the technical field of biological medicines. The invention takes a pH response type asymmetric cyanine structure as a core unit, and constructs photo-thermal/photodynamic/fluorescent integrated probe molecules which can be specifically activated by a tumor weak acid microenvironment through structural modification and introduction of heavy atoms such as halogen in a conjugated system. The photo-thermal/photodynamic/fluorescent integrated probe has good stability, photo-thermal effect and excellent capability of generating active oxygen, and has great potential in the aspect of tumor treatment.)

1. A compound used as a pH response type photo-thermal/photodynamic/fluorescent integrated probe molecule has a structure shown in a formula (I):

wherein R is₁、R₂Are respectively selected from the following stituents: hydrogen, nitro, amino, bromine,

2. A process for preparing a compound of claim 1, comprising the steps of:

(1) taking the compound 1 and 3-methyl-2-butanone as starting materials, and reacting to obtain a compound 2 and a compound 2';

(2) reacting the compound 2 serving as an initiator with methyl 4-bromomethylbenzoate to obtain a compound 3;

(3) taking the compounds 2,3 and 4 as reaction substrates, and reacting to obtain a compound shown in a formula (I);

wherein, compound 1:compound 2:the compound 2':compound 3:compound 4:R、R₁、R₂selected from: h, Br, NO₂，NH₂，

3. The method according to claim 2, wherein in step (1), the molar ratio of compound 1 to 3-methyl-2-butanone is 1: (1-2); the reaction temperature is 80-120 ℃, and the reaction time is 2-3 h.

4. The process of claim 2, wherein in step (2), the molar ratio of compound 2 to methyl 4-bromomethylbenzoate is 1: (1-2); the reaction temperature is 80-120 ℃, and the reaction time is 4-6 h.

5. The method of claim 2, wherein the preparing process of step (3) comprises: the molar ratio of compounds 2,3,4 was 1: 1: 1, heating to 50 ℃ for reaction for 5 hours under the protection of nitrogen by using N, N-dimethylformamide as a solvent.

6. A pH-responsive photothermal/photodynamic/fluorescent integrated probe molecule, wherein the pH-responsive photothermal/photodynamic/fluorescent integrated probe molecule is a compound having a structure represented by the formula (1) in claim 1, wherein R is₁、R₂Are each a substituent as defined in claim 1.

7. Use of the compound of claim 1 or the pH-responsive photothermal/photodynamic/fluorescent probe molecule of claim 6 for preparing a photothermal/photodynamic/fluorescent diagnostic agent.

8. Use of the compound of claim 1 or the pH-responsive photothermal/photodynamic/fluorescent integrated probe molecule of claim 6 in the field of bioimaging for non-disease diagnosis and therapy.

9. Use of the compound of claim 1 or the pH-responsive photothermal/photodynamic/fluorescent integrated probe molecule of claim 6 for the preparation of a medicament for the treatment of tumors.

10. The use of claim 9, wherein the medicament further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a pH reversible activation type photo-thermal/photodynamic/fluorescent integrated probe molecule.

Background

The early diagnosis and treatment of cancer is the key for improving the cure rate of cancer patients, and the development of a detection technology with strong specificity and high sensitivity for early diagnosis of tumors is very necessary. The optical imaging technology has many advantages of non-invasiveness, real-time performance, high sensitivity, simple operation, strong visibility and the like, and becomes an ideal method for detecting tumors. Especially, the near infrared fluorescence imaging technology is favored because the absorption and emission wavelengths are both in the biological optical imaging window, and has the advantages of strong tissue penetration capability, small light absorption and autofluorescence interference of biological tissues, and the like. However, most of the traditional imaging probes are always 'always on' type developers, and the development always in a fluorescence activation state always shows fluorescence signals in the metabolic transfer process in vivo, so that the problems of self background interference, false positive results and the like are easily caused.

The phototherapy is a mild, local and relatively safe treatment mode, has no obvious biotoxicity, generates a specific killing effect under the irradiation of external light, and shows good application prospect in the aspect of accurate treatment of tumors. Depending on the difference of light energy converted into heat energy or active oxygen after the light-heat/photosensitizer is irradiated by external light, the light-heat/photosensitizer can be classified into Photothermal therapy (PTT) and Photodynamic therapy (PDT). In recent years, a combination therapy has been reported by combining novel therapies such as PDT and PTT, and a better therapeutic effect than that of the single therapy has been achieved. However, most of the photosensitizers used for photothermal/photodynamic therapy in the existing research are combinations of two different materials, and most of the photosensitizers are always on type photothermal/photosensitizers, and due to the difference of physicochemical properties of the photosensitizers, the two materials are easily distributed in vivo and the selection of a target area is not completely the same, so that the real and accurate combined therapy is difficult to realize; or exciting light stimulation with two wavelengths is needed, so that the treatment time is prolonged, and side effects and operation difficulty are increased; meanwhile, non-specific damage to normal tissues cannot be avoided.

Disclosure of Invention

In order to solve the problems, a novel probe molecule integrating the specific activation type of a tumor microenvironment with functions of photothermal, photodynamic and fluorescence is designed and synthesized, and the probe molecule is used for photothermal/photodynamic combined therapy guided by accurate tumor imaging. The invention provides a photo-thermal/photodynamic/fluorescent integrated probe molecule which can be specifically activated by a tumor weak acid microenvironment, a preparation method and application thereof, wherein a pH response type asymmetric cyanine structure is used as a core unit, and photo-thermal/photodynamic/fluorescent integrated probe molecules which can be specifically activated by the tumor weak acid microenvironment are constructed by structure modification and introduction of heavy atoms such as halogen in a conjugated system.

The technical scheme is as follows:

a compound used as a pH response type photo-thermal/photodynamic/fluorescent integrated probe molecule has a structure shown in a formula (I):

wherein R is₁、R₂Each independently selected from the following substituents: hydrogen, nitro, amino, bromine,

In one embodiment of the present invention, the method for preparing a pH-responsive photothermal/photodynamic/fluorescent integrated probe molecule comprises: the method comprises the following steps:

(1) a compound of formula 1:and 3-methyl-2-butanone as a starting material, and under the conditions of acetic acid and concentrated sulfuric acid, obtaining a compound 2:and compound 2':

(2) reacting compound 2 serving as an initiator with methyl 4-bromomethylbenzoate to obtain compound 3:

(3) reacting compounds 2', 3,4 to obtain the compound of formula (I):

wherein R is R₁Or R₂；R₁、R₂Are respectively and independently selected from H, Br and NO₂、NH₂、

In one embodiment of the present invention, the preparation method specifically includes the following steps:

step (1): the mol ratio of the compound 1 to the 3-methyl-2-butanone is 1: l-2, heating to 100 ℃, reacting for 2h, cooling to room temperature, extracting with ethyl acetate, concentrating, adding 20mL of glacial acetic acid into the obtained red liquid, refluxing for 5h, cooling to room temperature, adding 50mL of dichloromethane, neutralizing the glacial acetic acid with saturated sodium carbonate aqueous solution, extracting with dichloromethane, concentrating, and performing column chromatography separation to obtain a compound 2;

step (2): the molar ratio of the compound 2 to the methyl 4-bromomethylbenzoate is 1: stirring for 5h at the temperature of 1-2,100 ℃, cooling to room temperature, adding 30mL of NaOH solution into the reaction system, and stirring for 1h at the room temperature; extracting with dichloromethane, rotary evaporating, concentrating, eluting with petroleum ether, ethyl acetate (40): 1(V/V) column chromatography separation to obtain a compound 3;

and (3): the molar ratio of compounds 2', 3,4 is 1: 1: 1, heating N, N-dimethylformamide serving as a solvent to 50 ℃ under the protection of nitrogen, and reacting for 5 hours. After cooling to room temperature, washing with water, extracting with dichloromethane, concentrating, and separating by column chromatography, wherein the eluent is petroleum ether, ethyl acetate: 1(V/V), followed by petroleum ether, ethyl acetate ═ 2:1(V/V) to obtain the compound of the formula (I).

In one embodiment of the present invention, the preparation method of the compound 4 comprises the following processes: firstly, cooling N, N-dimethylformamide to below 0 ℃ in an ice salt bath, dropwise adding a mixed solution of phosphorus oxychloride and dichloromethane, and continuously stirring for 0.5 h; slowly adding cyclohexanone dropwise, removing the ice bath after dropwise adding, heating the reaction solution to 80 ℃, reacting for 4 hours, cooling to room temperature, and washing with acetone to obtain a compound 4; wherein the molar ratio of the phosphorus oxychloride to the cyclohexanone is 1: (1-2).

The second purpose of the invention is to provide a pH response type photo-thermal/photodynamic/fluorescent integrated probe molecule, which is a compound with a structure shown in a formula (1), wherein R is₁、R₂Are each a substituent as defined in the formula (1).

The third purpose of the invention is to apply the compound or the pH response type photo-thermal/photodynamic/fluorescent integrated probe molecule in the field of biological imaging.

It is a fourth object of the present invention to provide a method for living cell imaging or in vivo imaging, which uses the above-mentioned compound as a probe. The cells may specifically include cancer cells such as lung cancer cell A549, mouse squamous cell carcinoma cell SCC-7, and the like.

The fifth purpose of the invention is to use the compound in preparing the medicine for treating tumor.

In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment of the invention, the dosage form of the medicament comprises injection, freeze-dried powder for injection, controlled release injection, liposome injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.

In one embodiment of the invention, the drug carrier includes microcapsules, microspheres, nanoparticles, and liposomes.

In one embodiment of the invention, the pharmaceutical excipients comprise solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.

In one embodiment of the invention, the pharmaceutical excipients comprise microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

The invention has the following remarkable advantages:

the invention takes an asymmetric cyanine structure with pH response performance as a core unit, and constructs photo-thermal/photodynamic/fluorescent integrated probe molecules which can be specifically activated by a tumor weak acid microenvironment through structural modification and introduction of heavy atoms such as halogen in a conjugated system.

The probe molecular compound can be activated by the specificity of a tumor weak acid microenvironment, so that the specificity of tumor diagnosis is improved; has the combined treatment effect of photo-thermal/photodynamic; the target compound has a single structure, no isomer exists, and the product is easy to purify.

The photo-thermal/photodynamic/fluorescent integrated probe with the reversibly activated pH has a novel molecular structure, has pH response performance, can effectively avoid interference of biological autofluorescence and cell endogenous substances by weak-acid microenvironment activated near-infrared absorption and emission, has strong specificity, high sensitivity and good optical stability, and can be used as a probe for detecting biological imaging and photo-thermal/photodynamic therapy. The fluorescent molecular probe has strong practical application value in the fields of analytical chemistry, life science and the like.

Drawings

FIG. 1 shows probe molecules obtained in example 1¹H NMR spectrum;

FIG. 2 is a mass spectrum of the probe molecule obtained in example 1;

FIG. 3 is a graph showing the UV absorption spectrum and fluorescence emission spectrum of the probe molecule obtained in example 1 at different pH values;

FIG. 4 is a graph showing photothermal effects of the probe molecule obtained in example 1;

FIG. 5 is a singlet oxygen evolution diagram of the probe molecule obtained in example 1;

FIG. 6 is a graph showing cytotoxicity of the probe molecules obtained in example 1;

FIG. 7 is a graph showing the in vitro cancer cell killing effect of the probe molecules obtained in example 1.

Detailed Description

The preparation method of the pH response type photo-thermal/photodynamic/fluorescent integrated probe molecule comprises the following steps:

in the step (1), the molar ratio of the compound 1 to the 3-methyl-2-butanone is 1: l-2, heating to 100 ℃, reacting for 2h, cooling to room temperature, extracting with ethyl acetate, concentrating, adding 20mL of glacial acetic acid into the obtained red liquid, refluxing for 5h, cooling to room temperature, adding 50mL of dichloromethane, neutralizing the glacial acetic acid with saturated sodium carbonate aqueous solution, extracting with dichloromethane, concentrating, and performing column chromatography separation to obtain the compound 2 with the yield of 78-82%.

The preparation process of the step (2) comprises the following steps: the molar ratio of the compound 2 to the methyl 4-bromomethylbenzoate is 1: stirring at 1-2,100 ℃ for 5h, cooling to room temperature, adding 30mL of NaOH solution into the reaction system, and stirring at room temperature for 1 h. Extracted with dichloromethane and concentrated by rotary evaporation. Separating by column chromatography, eluting with petroleum ether, and then petroleum ether: ethyl acetate 40:1(V/V) gives the compound 3 in 70-75% yield.

The preparation process of the step (3) comprises the following steps: the molar ratio of the phosphorus oxychloride to the cyclohexanone is 1: 1-2, adding N, N-dimethylformamide into a 250mL two-mouth bottle, magnetically stirring, cooling to below 0 ℃ in an ice salt bath, dropwise adding a mixed solution of phosphorus oxychloride and dichloromethane, and continuously stirring for 0.5 h. Then slowly adding cyclohexanone dropwise, removing the ice bath after dropwise adding, heating the reaction solution to 80 ℃, and reacting for 4 hours. Cooling to room temperature and washing with acetone gave compound 4 in 85-89% yield.

The preparation process of the step (4) comprises the following steps: the molar ratio of compounds 2,3,4 was 1: 1: 1, heating N, N-dimethylformamide serving as a solvent to 50 ℃ under the protection of nitrogen, and reacting for 5 hours. Cooling to room temperature, washing with water, extracting with dichloromethane, concentrating, and separating by column chromatography, wherein the eluent is petroleum ether, ethyl acetate, 10: 1(V/V) followed by petroleum ether ethyl acetate ═ 2:1(V/V) to give the compound of formula (I) in 33-40% yield.

The application performance evaluation of the pH response type photo-thermal/photodynamic/fluorescent integrated probe comprises the following steps:

the main research contents comprise the evaluation of the optical property, the photothermal conversion capability, the singlet oxygen generating capability, the cytotoxicity and the cell killing effect of the probe.

Optical characterization of the molecules: accurately weighing probe molecules to prepare the probe molecules with the concentration of 10^-3M ethanol stock solution. The solvent used is ethanol: water 1: 1 (volume ratio) dilution of 10^-5M, then adjusting the pH of the solution to be tested to 7.4 (simulating normal body fluid) and 6.0 (simulating tumor weak acid microenvironment) by using NaOH (1M) and HCl (1M) solutions respectively. The absorption and emission spectra (excitation wavelength is the maximum emission wavelength of the probe) were measured with an ultraviolet spectrophotometer and a fluorescence spectrometer, respectively.

Measuring photothermal conversion ability by taking 0.8mL of the above solution (1 × 10)^-5M) (pH 6.0 and 7.4, respectively) were placed in 1.5mL centrifuge tubes at different powers (0.2, 0.4, 0.6, 0.8 and 1.0w cm^-2) Irradiating for 10min by a 808nm laser, collecting thermal imaging pictures by a thermal imager at intervals of 30s, recording the temperature, and drawing a change curve of the temperature along with the time.

Measuring the singlet oxygen generating capacity, taking the concentration of 19.9mL as 6 × 10^-5DPBF solution of M and 100uL concentration of 10^-3M was mixed in a 50mL centrifuge tube and shaken well. Duplicate samples were adjusted to pH 7.4 and 6.0, and wrapped in tinfoil paper and stored in dark. The test solution was dispensed into 1.5mL centrifuge tubes (1 mL per tube). The power consumption is 0.6w cm^-2For different times (0s, 10s, 20s, 30s, 40s, 50s, 1min, 1.5min, 2min, 3min and 5 min). The absorbance of the irradiated solution was measured with a UV-Vis spectrophotometer.

Cytotoxicity assay (determination of cell viability MTT method) biocompatibility determination was carried out by MTT method briefly, 1 × 10 was added to a 96-well plate⁵cell/mL cell suspension (100. mu.L/well) and grown overnight, then the medium was removed and 100. mu.L of probe (2 × 10) containing varying concentrations was added^-6,5×10^-6，8×10^-6，1×10^-5M) in fresh medium, 5 replicates per group, and incubation continued for 24 hours. The medium was removed, washed with PBS, and 100. mu.L of 0.5mg mL solution was added^-1MTT in fresh medium, and culturing for 4 h. The well was aspirated, washed once with PBS, added with 100. mu.L DMSO, and shaken in a constant temperature shaker at 37 ℃ for 30min in the dark. Measuring the absorbance at a wavelength of 570nm with a microplate reader (A)₅₇₀). The cell viability calculation formula is as follows: cell viability test group A₅₇₀Control group A₅₇₀×100％。

Evaluation of cell photothermal/photodynamic killing effect: cell culture medium incubation was performed as described above for cytotoxicity assays. Adjusting the pH value of the culture system to 6.0 or 7.4 after the cells and the probes are incubated, irradiating for 10min by using lasers with different powers and then incubating for 12 h. After 12h, wash with PBS once and replace with 100uL of 0.5mg mL^-1Fresh medium of MTT was cultured for an additional 4 h. Remove the liquid in the wells, wash with PBS, add 100uL DMSO, shake in a constant temperature shaker at 37 ℃ for 30min in the dark. The absorbance at a wavelength of 570nm was measured with a microplate reader.

The invention is further illustrated by the following examples, but is not limited thereto.