阅读说明：本技术 一种偶氮苯/cb[7]/聚多巴胺复合物制备及在光热肿瘤治疗方面的应用 (Preparation of azobenzene/CB [7 ]/polydopamine compound and application in photothermal tumor treatment ) 是由 程红波 文簙锌 赵静 胡陈燕 于 2021-07-20 设计创作，主要内容包括：一种偶氮苯/CB[7]/聚多巴胺复合物制备及在光热肿瘤治疗方面的应用,属于生物医药技术领域。吡啶离子化的偶氮苯化合物Azo结构式如下：将4,4’-二羟基偶氮苯溶解后加入1,3-二溴丙烷,在碳酸钾的作用下反应合成4,4’-二(3-溴丙氧基)偶氮苯,该物质经纯化后继续与吡啶反应得到吡啶离子化的偶氮苯化合物Azo。然后制备正电荷偶氮苯与CB[7]的组装体,最后加入多巴胺进行聚多巴胺的包覆,得到偶氮苯/CB[7]/聚多巴胺复合物。葫芦脲与偶氮苯分子的组装形貌,并以该形貌为核心,原位负载上聚多巴胺,使其具有良好的光热性能,达到治疗肿瘤的目的。(Azobenzene/CB [7]]Preparation of polydopamine compound and application thereof in photothermal tumor treatment, belonging to the technical field of biological medicine. The pyridine-ionized azobenzene compound has the following structural formula:)

1. A pyridine-ionized azobenzene compound Azo, characterized in that the compound has the following structural formula:

2. the process for producing an Azo-benzene compound (Azo) with ionized pyridine according to claim 1, comprising the steps of:

dissolving 4,4 '-dihydroxyazobenzene, adding 1, 3-dibromopropane, reacting under the action of potassium carbonate to synthesize 4,4' -di (3-bromopropoxy) azobenzene, purifying the substance, and continuing to react with pyridine to obtain pyridine-ionized azobenzene compound Azo.

3. A method for preparing an assembly of positively charged azobenzene and CB [7], comprising the steps of:

firstly, preparing Tris-hydrochloric acid buffer solution, adjusting the pH value to 7-9, optimally to pH8.5 by using sodium hydroxide solution, adding 1eq to pyridine ionized azobenzene compound Azo into the solution system for fully dissolving, then adding 0.5-1.5eq (preferably 1eq) cucurbituril [7], shaking up, and carrying out ultrasonic treatment for 30 minutes to obtain an assembly of the positive charge azobenzene and CB [7] with fibrous morphology.

4. An assembly of positively charged azobenzene and CB [7] prepared by the process of claim 3.

5. A method for preparing positive charge azobenzene/CB [7 ]/polydopamine compound is characterized in that the dopamine adhered on the fibrous assembly is gradually polymerized under alkaline environment by utilizing the adhesiveness of the dopamine; the method comprises the following specific steps:

firstly, preparing a Tris-hydrochloric acid buffer solution, adjusting the pH value to 7-9, optimally to 8.5 by using a sodium hydroxide solution, adding 1eq of pyridine ionized azobenzene compound into the solution system, carrying out Azo full dissolution, then adding 0.5-1.5eq (preferably 1eq) of cucurbituril [7], shaking up, and carrying out ultrasonic treatment for 30 minutes; adding 0.5-3eq (preferably 2eq) of dopamine into the solution, stirring with magnetons, stirring at room temperature for at least 5 hours, finishing the reaction, centrifuging the reaction system, and removing the supernatant until the supernatant is not obviously brown; it is subsequently ultrafiltered on an ultrafiltration tube (preferably 100kD at 10000 rpm), the supernatant is discarded and the product is washed with deionized water.

6. A positively charged azobenzene/CB [7 ]/polydopamine complex prepared according to the process of claim 5.

7. The use of the positively charged azobenzene/CB [7 ]/polydopamine complexes prepared according to the process of claim 5 as photothermal materials.

8. Use of a positively charged azobenzene/CB [7 ]/polydopamine complex prepared according to the process of claim 7 in the preparation of a reagent or device for the treatment of tumours.

Technical Field

The invention relates to a preparation method and application of an assembly body biological photo-thermal material, in particular to application of polydopamine to tumor photo-thermal treatment, and belongs to the technical field of biological medicines.

Background

With the continuous development of biomedicine, more and more diseases are discovered, and in recent years, the incidence rate of tumors is higher, so that the diagnosis and treatment of tumors become a great problem in the world. At present, a plurality of methods are used for treating tumors, such as operation, chemotherapy, radiotherapy and the like, but the treatment effect and the brought side effect of the methods still cause headache, the application of the nano material to photothermal treatment is a promising treatment means, and meanwhile, most photothermal materials can perform photoacoustic imaging, so that the treatment visualization of tumor parts is facilitated.

Supramolecular chemistry is a new chemical branch which is emerging at the end of the 20 th century, and the supramolecular chemistry is firstly formed on the basis of subject-object chemistry proposed by scientists such as Leign and the like, and then supplemented by Stoddart, Fernga and the like, so that a large important research subject covering various intermolecular actions and intermolecular mechanical interlocking mechanisms is formed at present, and the concept of the supramolecular chemistry is widely applied to various fields such as material chemistry, biochemistry, environmental chemistry and the like. Host-guest chemistry is the most important branch in the field of supramolecular chemistry, and mainly studies the interaction mechanism between a host with a cavity-containing structure and a guest with a proper molecular scale. At present, the most mature host-guest interaction mechanism is a macrocyclic chemical interaction mechanism, mainly comprising the interlocking and embedding of a large-ring capsule cavity structure such as cyclodextrin, cucurbituril, calixarene and the like and a plurality of small molecules, and the action modes provide new ideas and directions for the design of a plurality of intelligent materials.

On the basis of the two backgrounds, the biocompatibility and the photo-thermal stability of the material are improved by selecting a poly-dopamine coating method on the surface of an organic whole assembled by supermolecules, and the material with a good photo-thermal effect can be obtained by the simple and easy-to-operate method.

Disclosure of Invention

The invention aims to provide a method for processing and preparing a composite photo-thermal material by using a supramolecular assembly as a matrix and application thereof.

An object of the present invention is to provide a pyridine-ionized azobenzene compound Azo and a method for preparing the same.

Another object of the present invention is to provide an assembly of positively charged azobenzene and CB 7 and a method for preparing the same.

It is a third object of the present invention to provide a positively charged azobenzene/CB < 7 >/polydopamine forming complex. Preparation and application thereof.

The method is realized by the following method technologies:

a pyridine-ionized azobenzene compound Azo, characterized in that the compound has the following structural formula:

a method for producing an azobenzene compound (Azo) having pyridine ionization, characterized by comprising the steps of:

dissolving 4,4 '-dihydroxyazobenzene, adding 1, 3-dibromopropane, reacting under the action of potassium carbonate to synthesize 4,4' -di (3-bromopropoxy) azobenzene, purifying the substance, and continuing to react with pyridine to obtain pyridine-ionized azobenzene compound Azo.

The invention provides a preparation method of an assembly of positive charge azobenzene and CB [7], which comprises the following steps:

firstly, preparing Tris-hydrochloric acid buffer solution, adjusting the pH value to 7-9, optimally to pH8.5 by using sodium hydroxide solution, adding 1eq to pyridine ionized azobenzene compound Azo into the solution system for fully dissolving, then adding 0.5-1.5eq (preferably 1eq) cucurbituril [7], shaking up, and carrying out ultrasonic treatment for 30 minutes to obtain an assembly of the positive charge azobenzene and CB [7] with fibrous morphology.

The invention relates to a method for preparing positive charge azobenzene/CB 7/polydopamine compound, which uses the adhesiveness of dopamine to polymerize the dopamine on the fiber assembly in alkaline environment; the method comprises the following specific steps:

firstly, preparing a Tris-hydrochloric acid buffer solution, adjusting the pH value to 7-9, optimally to 8.5 by using a sodium hydroxide solution, adding 1eq of pyridine ionized azobenzene compound into the solution system, carrying out Azo full dissolution, then adding 0.5-1.5eq (preferably 1eq) of cucurbituril [7], shaking up, and carrying out ultrasonic treatment for 30 minutes; adding 0.5-3eq (preferably 2eq) of dopamine into the solution, stirring with magnetons, stirring at room temperature for at least 5 hours, finishing the reaction, centrifuging the reaction system, and removing the supernatant until the supernatant is not obviously brown; it is subsequently ultrafiltered on an ultrafiltration tube (preferably 100kD at 10000 rpm), the supernatant is discarded and the product is washed with deionized water. Meanwhile, the thickness of the outer poly-dopamine shell layer of the assembly body can be controlled by changing the dosage of the monomer dopamine, the pH value of the synthetic solution and the time for oxidizing and self-polymerizing the monomer dopamine, so that the size of the material is changed.

The invention aims to provide a novel assembling morphology of cucurbituril and azobenzene molecules, and the morphology is used as a core, and polydopamine is loaded in situ, so that the cucurbituril molecules have good photo-thermal properties, and the purpose of treating tumors is achieved, and therefore, the cucurbituril molecules can be used as reagents or devices for preparing the agents for treating the tumors.

The invention has the advantages that:

the poly-dopamine-coated photothermal reagent synthesized by the method has the advantages that the poly-dopamine can be synthesized in a human body, so that the compatibility of the poly-dopamine and the human body is good, the material has the characteristics of good biocompatibility, excellent photothermal effect and the like, the poly-dopamine has good absorption in near infrared, a good photothermal effect can be generated under the excitation of 808nm, the temperature can be raised to about 60 ℃ from room temperature within 3min, and the poly-dopamine-coated photothermal reagent has the potential of changing the morphological structure due to the unique morphological structure, for example, reported glutathione in the body can peel off the poly-dopamine, and the exposed fibrous assembly can activate the in vivo immunity, so that the effect of killing cancer cells under the autoimmunity is achieved.

Drawings

FIG. 1a is a schematic diagram of Azo benzene compound Azo structure with pyridine ionization, b is a schematic diagram of cucurbituril [7], and c is a schematic diagram of structure of Azo and cucurbituril [7] assembly.

FIG. 2 is a nuclear magnetic spectrum of 2mmol of azobenzene at the upper part and a nuclear magnetic spectrum of an assembled structure of Azo and cucurbituril [7 ];

FIG. 3a is an Azo ultraviolet absorption diagram, b is an Azo and cucurbituril assembly ultraviolet absorption diagram, and c is an ultraviolet absorption diagram of the polydopamine-loaded polymer synthesized by the assembly under different pH values;

FIG. 4 is a photothermal curve of assembly loading with polydopamine;

FIG. 5a is a TEM image of the Azo and cucurbituril [7] assembly, and b is a TEM image of the assembly loaded with polydopamine.

Detailed Description

The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.

Example 1

Preparation of pyridine-ionized azobenzene compound Azo: dissolving 4,4 '-dihydroxyazobenzene 1eq in 100mL THF, adding 1eq.1, 3-dibromopropane, adding 1 eq.potassium carbonate, heating the reaction mixture under reflux for 24h, removing the solvent by rotary evaporation, purifying with a column to obtain 4,4' -bis (3-bromopropoxy) azobenzene, dissolving the product in 30mL DMF, adding 1 eq.pyridine dropwise, heating to 90 deg.C under reflux for 24h, filtering the precipitate and washing with acetonitrile. The solid was completely dissolved in a small amount of deionized water and concentrated NH was added₄PF₆Aqueous solution until no further precipitation was observed, the precipitate was filtered off and washed with water to give hexafluorophosphate. Exchange the counter ion with tetramethylammonium bromide to Br^-The yellow solid gave the pyridine-ionized azobenzene compound Azo.

5 to 12mg, preferably 8mg of the pyridine-ionized azobenzene compound was dissolved in 0.5mL of heavy water and subjected to a nuclear magnetic resonance hydrogen spectroscopy (H NMR) test, as shown in the upper graph of FIG. 2, which corresponds to the Azo. The total 8 groups of peaks (a-h) are found, which respectively correspond to a-h groups of hydrogen in the molecular structure, and after integration, the integral area ratio of the peaks is basically consistent with the corresponding hydrogen quantity ratio. It was also found that azobenzene molecules spontaneously interconvert in aqueous solution to give structures to the right of 2 corresponding to Azo, with hydrogen chemical shifts corresponding to e ', g ', h ' in the figure, respectively.

Example 2

Preparation of an Azo and CB [7] assembly: firstly, preparing Tris-hydrochloric acid buffer solution, using sodium hydroxide solution to regulate pH value to 8.5, adding 1eq of pyridine ionized azobenzene compound into said solution system, and fully dissolving, then adding 1eq cucurbituril [7] namely guanidine 7, shaking up, ultrasonic treating for 30 min so as to obtain the fibrous assembly of Azo and CB [7 ].

The assembly of 5 to 12mg, preferably 8mg of pyridine-ionized azobenzene compound Azo and guanidine 7 was dissolved in 0.5mL of heavy water and subjected to nuclear magnetic resonance hydrogen spectroscopy (H NMR) test, as shown in the lower graph of FIG. 2. It is found that 11 groups of peaks (a-h, A-C) are appeared together, and the integrated area ratio of the groups of the hydrogen in the a-h in the azobenzene molecular structure and the groups of the hydrogen in the A-C in the guanidine 7 molecule are basically consistent with the corresponding hydrogen quantity ratio after integration. The ratio of guanidine 7 to azobenzene was about 1.4:1, with a slight excess of guanidine 7. Compared with an H NMR spectrum of azobenzene, the chemical shift of the a-c three groups of peaks in the supramolecular assembly is remarkably increased, and the chemical shift of the f-H three groups of peaks is remarkably reduced, which is consistent with the effect result of guanidine 7 on pyridine rings and carbon chains, and shows that the supramolecular complex is formed.

Taking 3mL of 30-mu M aqueous solution of Azo, and obtaining an absorption curve shown as a in figure 3 by an ultraviolet absorption test; 30 μ M of the assembly, namely Azo and guanidine [7]]Is 1: 1 Assembly of guanidine [7] to ensure complete Azo assembly]Slightly excessive, 3mL of the assembly aqueous solution, the absorption curve obtained by the assembly ultraviolet absorption test is shown as b in fig. 3, the ultraviolet curve is subjected to Fourier peak separation by origin to obtain three peaks, and the positions of the ultraviolet absorption peaks are analyzed, so that the absorption at 314nm in a in fig. 3 corresponds to a benzene ring, the relative peak height is 0.27, and the peak area is 31.3; absorption at 361nm corresponds to azo bonds, phaseThe peak height was 0.26, and the peak area was 35.5; the absorption at 413nm corresponds to pyridine, the relative peak height is 0.12, and the peak area is 33.2; the absorption positions, relative peak heights and peak areas of the three chemical groups in b in the graph in FIG. 3 are 316nm, 0.24 and 27.7 respectively; 365. 0.20, 22.3; 397. 0.18, 49.9. By comparison, it was found that the peak of the pyridine ring was enhanced and the benzene ring and azo bond were suppressed, indicating that the pyridine ring was wrapped with azobenzene to form an assembly. In FIG. 3 c is the above-mentioned UV absorption spectrum of the assembly of FIG. 3a, b and three different pH values of 7.5, 8.0 and 8.5 at a concentration of 200. mu.g/mL, wherein the most preferred UV absorption spectrum of 8.5 is shown at pH8.5The absorption value of @ PDA is the largest, and the absorption of the @ PDA is wide from 200nm to 900nm, which also proves the photo-thermal potential of the @ PDA.

Example 3

Azobenzene/CB [7]]Preparation of Polydopamine-forming pseudorotaxane complexes: in example 2Azo and CB [7]]Adding 2eq of dopamine into the solution of the assembly, stirring with a magneton, stirring at room temperature for at least 5 hours, finishing the reaction, centrifuging the reaction system, and removing the supernatant until the supernatant is not obviously brown; subsequently, it is ultrafiltered with an ultrafiltration tube (preferably 100kD at 10000 rpm), the supernatant is discarded, and the resultant is washed with deionized water to obtain a solution having a product pH of 8.5]Polydopamine complexes as@ PDA complex, and freeze-drying to obtainThe @ PDA complex was prepared as a powdery solid in a 500. mu.g/mL solution, and 1mL of the assembly solution was taken and the applied power was 1W/cm²The 808 laser irradiates a solution system for 3min, the temperature is tested every 30s, and the highest energy of the system is increased to about 60 ℃ within 3min, which shows that the assembly has good photo-thermal performance.

As shown in FIG. 5, at 30 μ MPreparing a transmission electron microscope observation sample by the assembly system, taking a drop on a 300-mesh copper net, sucking away excessive liquid by using filter paper, rinsing twice by using clear water, observing to obtain a fibrous photo shown in a figure 5 after sample preparation is finished, and preparing the fiber photo by the same methodThe TEM of @ PDA was observed to obtain a TEM photograph as shown in B in FIG. 5.