阅读说明：本技术 一种功能化NH2-MIL125(Ti)及其制备方法和应用 (Functional NH2-MIL125(Ti) and preparation method and application thereof ) 是由 舒细记 商锦婷 王海萍 孙宾莲 柳威 王磊 刘钰晨 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种功能化NH-2-MIL125(Ti)及其制备方法和应用,其制备方法包括NH-2-MIL125(Ti)的合成、重氮化染料苯并三氮唑的合成,以及采用所述重氮化染料苯并三氮唑对NH-2-MIL125(Ti)进行染料敏化,获得了功能化NH-2-MIL125(Ti),并进一步发现该功能化NH-2-MIL125(Ti)在450nm单波长光激发下对于癌细胞尤其是口腔鳞癌细胞的杀灭效果显著增强,因此可将其制备成口腔鳞癌细胞治疗药物并应用于临床中,同时本发明提供的该功能化NH-2-MIL125(Ti)的制备方法简单快速,适合大规模生产使用。(The invention discloses a functional NH 2 -MIL125(Ti) and its preparation method and application, the preparation method comprises NH 2 -MIL125(Ti) synthesis, diazotised dye benzotriazole synthesis, and use of said diazotised dye benzotriazole for the treatment of NH 2 MIL125(Ti) for dye sensitization to obtain functionalized NH 2 MIL125(Ti), and further finding the functionalized NH 2 The killing effect of MIL125(Ti) on cancer cells, particularly oral squamous carcinoma cells, is remarkably enhanced under the excitation of 450nm single-wavelength light, so that the MIL125(Ti) can be prepared into the oral squamous carcinoma cellsThe cell therapy medicine is applied to clinic, and the functionalized NH provided by the invention 2 The preparation method of-MIL 125(Ti) is simple and rapid, and is suitable for large-scale production and use.)

1. Functional NH₂-method for preparing MIL125(Ti), characterized in that it comprises:

step 1, NH₂Synthesis of MIL125 (Ti): adding 2-amino terephthalic acid into a mixed solution of dimethyl formamide and methanol, then adding isopropyl titanate, ultrasonically mixing, transferring into polytetrafluoroethylene, reacting at 150 ℃ for 72h, cooling to room temperature, washing and separatingDrying the core to obtain NH₂-MIL125(Ti)；

Step 2, synthesis of diazotization dye benzotriazole: dissolving 4- (6-methyl-1, 3-benzothiazole-2-yl) aniline in ice water, adding hydrochloric acid, then dripping sodium nitrite, continuously stirring for reacting for 1-2h, then adding fluoboric acid, shaking for reacting for 1-2h, filtering, washing and drying to obtain diazotized dye benzotriazole;

step 3, adding the NH₂Mixing MIL125(Ti), the diazotized dye benzotriazole and tetrabutyl hexafluorophosphate, adding into acetonitrile, reacting in dark environment under the protection of nitrogen, centrifuging and removing supernatant to obtain the functionalized NH₂-MIL125(Ti)。

2. The method according to claim 1, wherein the NH in step 3₂The mass ratio of-MIL 125(Ti) to diazotized dye benzotriazole is 4: 1.

3. The method according to claim 1, wherein the reaction in step 3 is stirred under nitrogen in a dark environment for 24 hours.

4. The method according to claim 1, wherein the functionalized NH is obtained by centrifuging the supernatant in step 3 to obtain a precipitate, and washing the precipitate three times with acetonitrile and water in sequence₂-MIL125(Ti)。

5. The method according to claim 1, wherein the volume ratio of dimethylformamide to methanol in step 1 is 9: 1.

6. Functional NH₂-MIL125(Ti), characterized in that it is obtained by the preparation process according to any one of claims 1 to 5.

7. The functionalized NH of claim 6₂Application of MIL125(Ti) in preparation of medicine for killing oral squamous cell carcinoma.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to functionalized NH₂MIL125(Ti) and a preparation method and application thereof.

Background

As a novel crystalline porous solid material, a metal-organic framework (MOFs) compound is formed by bridging metal ions or cluster compounds serving as nodes and organic ligands, has the characteristics of high specific surface area, multiple active sites, clipping property, functionalization and the like, and is widely concerned in the field of photocatalysis. MIL-125(Ti) is an important MOFs material. In recent years, MOFs have outstanding advantages in the field of photocatalysis, especially in the visible light degradation of pollutants, through the introduction of NH₂The functionalized ligand has no influence on the structure, and can expand the visible light absorption range, further increase the photocatalytic performance, and obviously improve the utilization efficiency of visible light.

Dye sensitization refers to the phenomenon that some organic dyes that are energy-matched with the conduction band and the valence band of a wide-bandgap semiconductor adsorb onto the surface of the semiconductor, and the strong absorption of visible light by the organic dyes is utilized to extend the spectral response of the system to the visible region.

Disclosure of Invention

The invention aims to provide a functional NH₂-MIL125(Ti) and preparation method and application thereof, by using diazotized dye benzotriazole to NH₂MIL125(Ti) for dye sensitization to obtain functionalized NH₂MIL125(Ti), and further finding the functionalized NH₂MIL125(Ti) has a remarkable killing effect on cancer cells, especially oral squamous carcinoma cells under the excitation of 450nm single-wavelength light.

In order to achieve the purpose, the invention adopts the technical scheme that:

functional NH₂-a method for preparing MIL125(Ti), comprising:

step 1, NH₂Synthesis of MIL125 (Ti): adding 2-amino terephthalic acid into a mixed solution of dimethyl formamide and methanol, then adding isopropyl titanate, ultrasonically mixing, transferring into polytetrafluoroethylene, reacting at 150 ℃ for 72 hours, cooling to room temperature, washing, centrifuging and drying to obtain NH₂-MIL125(Ti)；

Step 2, synthesis of diazotization dye benzotriazole: dissolving 4- (6-methyl-1, 3-benzothiazole-2-yl) aniline in ice water, adding hydrochloric acid, then dripping sodium nitrite, continuously stirring for reacting for 1-2h, then adding fluoboric acid, shaking for reacting for 1-2h, filtering, washing and drying to obtain diazotized dye benzotriazole;

step 3, adding the NH₂Mixing MIL125(Ti), the diazotized dye benzotriazole and tetrabutyl hexafluorophosphate, adding into acetonitrile, reacting in dark environment under the protection of nitrogen, centrifuging and removing supernatant to obtain the functionalized NH₂-MIL125(Ti)。

Further, NH in step 3₂The mass ratio of MIL125(Ti) to diazotized dye benzotriazole is.

Further, in step 3, the reaction is stirred for 24 hours in a dark environment under the protection of nitrogen.

Further, centrifuging in step 3 to remove supernatant to obtain precipitate, and washing the precipitate with acetonitrile and water sequentially for three times to obtain the functionalized NH₂-MIL125(Ti)。

Further, the volume ratio of the dimethylformamide to the methanol in the step 1 is 9: 1.

The invention also provides a functional NH₂MIL125(Ti), obtained by the above-mentioned preparation process.

The invention also provides the functional NH₂Application of MIL125(Ti) in preparation of medicine for killing oral squamous cell carcinoma.

Compared with the prior art, the invention has the beneficial effects that: the invention utilizes diazotizationDye benzotriazole p-NH₂MIL125(Ti) to obtain functionalized NH₂MIL125(Ti), and further finding the functionalized NH₂The killing effect of MIL125(Ti) on cancer cells, particularly oral squamous carcinoma cells, is remarkably enhanced under the excitation of 450nm single-wavelength light, so that the MIL125(Ti) can be prepared into an oral squamous carcinoma cell therapeutic drug and applied to clinic. The invention also provides the functional NH₂The preparation method of the MIL125(Ti) is simple and quick, and is suitable for large-scale production and use.

Drawings

FIG. 1 shows the functionalized NH prepared in example 1 of the present invention₂-characterization of MIL125(Ti), wherein fig. a is a TEM image and fig. B is a SEM image.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The present embodiment provides a functionalized NH₂Preparation method of-MIL 125(Ti) and verification of the functionalized NH₂-killing effect of MIL125(Ti) on oral squamous carcinoma cells.

The preparation method specifically comprises the following steps:

step 1, NH₂Synthesis of MIL125 (Ti): adding 0.5g of 2-aminoterephthalic acid into a mixed solution of 9mL of Dimethylformamide (DMF) and 1mL of methanol, then adding 0.26mL of isopropyl titanate, ultrasonically mixing for 30min, transferring the mixture into a reaction kettle containing polytetrafluoroethylene, reacting for 72h at 150 ℃, and cooling to room temperature to obtain a crude product NH₂-MIL125(Ti), washing with DMF and methanol respectively, centrifuging for 3 times, and drying in a vacuum drying oven at 120 deg.C for 24h to remove solvent to obtain the NH₂-MIL125(Ti)。

Step 2, synthesis of diazotized dye Benzotriazole (BTA): dissolving 80mg of 4- (6-methyl-1, 3-benzothiazole-2-yl) aniline in 100mL of ice water while stirring, dropwise adding hydrochloric acid until the mixture is completely dissolved, then dripping 50mg of sodium nitrite solution in the ice water mixed solution at the temperature of 0 ℃, continuously stirring for reaction for 1h, then adding 2mL of 50% fluoroboric acid solution and shaking for 1h, filtering to obtain a precipitate, washing with ice water and methanol for 3 times respectively, and drying to obtain the diazotized dye BTA;

step 3, taking 20mg of NH₂MIL125(Ti), 5mg of diazotized dye BTA and 11.6mg of tetrabutylammonium hexafluorophosphate ([ Bu)₄N]⁺PF₆ ^-) Mixing and dissolving in acetonitrile, reacting and stirring the mixed solution in a dark environment for 24 hours under the protection of nitrogen, centrifuging at 12000rpm for 10min after the reaction is finished, removing supernatant, washing with acetonitrile and water for 3 times in sequence after precipitates are obtained, and obtaining the functionalized NH₂-MIL125(Ti)。

Respectively taking the NH which is prepared in the step 1 and is not sensitized by the dye₂MIL125(Ti) and the functionalized NH₂MIL125(Ti), dispersed in PBS to a final concentration of 20. mu.g/mL, incubated with oral squamous cell carcinoma cells for 4h, irradiated with 455nm light for 2min, and examined for the survival of the oral squamous cell carcinoma cells in both groups using flow cytometry.

Cell experiments: the MTT method is a common method for detecting cell survival, the principle of the MTT method is that succinic dehydrogenase in living mitochondria can reduce exogenous MTT into water-soluble blue-violet crystal formazan and deposit the water-soluble blue-violet crystal formazan in cells, and the activity of the succinic dehydrogenase in dead cells is not enough to reduce MTT into the formazan, so that the content of the formazan in the cells is in direct proportion to the number of the living cells. Dimethyl sulfoxide can dissolve formazan in cells to form a purple solution, and an absorption value at 570nm is detected by a spectrophotometry method, so that the number of living cells is indirectly reflected.

Collecting oral squamous carcinoma cells, digesting with 0.25% trypsin, collecting cells, and adjusting cell density to 2 × 10 with corresponding cell culture solution⁵cells/mL, adjusted density cells were plated in 35mm dishes (1.4 mL/dish) and cells were randomly assignedControl group, administration illumination group, culturing for 12h, discarding supernatant, replacing fresh culture medium, mixing, inoculating into 96-well plate with each well having 100 μ L, each plate having 4 multiple wells, adding 5 μ g/mL NH into administration group and administration illumination group respectively₂MIL125(Ti) or dye-sensitized NH₂MIL125(Ti) is placed in an incubator and is protected from light to react for 4 hours, then the incubator is irradiated by LED with the wavelength of 450nm for 2 minutes, then is cultivated for 24 hours after serum is added, a 96-hole plate is taken out, supernatant is discarded, 150 microliter DMSO is added into each hole, the plate is placed on a flat shaking table to shake for 15 minutes, and the formazan generated by the MTT reaction is fully dissolved. The absorbance at 570nm (OD value) was measured using a microplate reader. The cell survival rate is the OD value of the experimental group/the control OD value of the normal group x 100%, and the experiment was repeated three times.

The results show that NH without dye sensitization is used₂After treatment of oral squamous carcinoma cells with MIL125(Ti), the viability of the cells was 90%, whereas with the functionalized NH₂After treatment of oral squamous carcinoma cells with MIL125(Ti), the survival rate of the cells was 46%. Namely the functionalized NH according to the invention₂MIL125(Ti) has obviously enhanced killing effect on oral squamous cell carcinoma cells under the excitation of light, so that the MIL125(Ti) can be prepared into an oral squamous cell carcinoma cell therapeutic drug and applied to clinic, and the invention functionalizes NH₂The preparation method of-MIL 125(Ti) is simple and rapid, and is suitable for large-scale production and use.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.