CN111840565A

CN111840565A - Fluorescent covalent organic framework nano-drug carrier and preparation method and application thereof

Info

Publication number: CN111840565A
Application number: CN202010757009.2A
Authority: CN
Inventors: 公培伟; 赵凯; 刘哲; 侯衍青; 许文宇; 张冉冉; 戚长敏
Original assignee: Qufu Normal University
Current assignee: Qufu Normal University
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-10-30

Abstract

The invention relates to a biological enzyme drug loaded on a covalent organic framework nano drug carrier, which is characterized in that a covalent organic framework material is obtained by reacting a monomer with N, N, N ', N' -tetra (4-formylphenyl) -1,1 '-biphenyl-4, 4' -diamine and a monomer with 3,3',5,5' -tetramethylbenzidine, and the covalent organic framework material has remarkable fluorescence property and is loaded with a glucose oxidase drug through adsorption. Simultaneously relates to a preparation method and application of the covalent organic framework nano-drug carrier. The carrier provides a convenient synthesis mode and a drug controlled release mode and a convenient route, can obviously improve the bioavailability of the drug, reduce the toxic and side effects of the drug and improve the treatment effect of the drug.

Description

Fluorescent covalent organic framework nano-drug carrier and preparation method and application thereof

Technical Field

The invention belongs to the technical field of materials and biomedicine, and particularly relates to a fluorescent covalent organic framework nano-drug carrier as well as a preparation method and application thereof.

Background

The development and use of anticancer drugs has been limited by the side effects of chemotherapeutic drugs. Although many anticancer drugs have a significant inhibitory effect on cancer cells, they also interfere with the functioning of normal cells and impose an excessive physical burden on patients, causing pain to the patients. The study of glucose oxidase in cancer therapy has well addressed this problem. It has little toxicity and side effects on normal cells due to the characteristics and specificity of its biological enzyme, but the process of delivering it to cancer cells is extremely difficult. Research on carriers of anticancer drugs poses new challenges. (see Y, Hu, H, Cheng, X.ZHao, ACS Nano, 11 (2017) 5558-2872.; J, Zhou, M, Li, Y, Hou, PhotothermalelTherapy, ACS Nano, 12 (2018)).

The appearance of covalent organic framework materials provides a new idea for solving the problems (see H. Wang, Z.Zeng, P. Xu, chem. Soc. Rev., 48 (2019) 488-doped 516; H. Wang, Z. Zeng, P. Xu, chem. Soc. Rev., 48 (2019)) and the materials not only have large conjugated structures and stronger covalent bonds, but also can be designed and synthesized by selecting different monomers according to requirements; meanwhile, the characteristics of the biological enzyme drug of the glucose oxidase loaded by the carrier enable the carrier to have the following advantages compared with other drug carriers:

the simpler design synthesis scheme: according to the previous experimental research and literature report, the experimental conditions of the covalent organic framework material designed and synthesized by us are not harsh, and the covalent organic framework material can be obtained at about 65-75 DEG CThe reaction was completed (see B. Wang, X. Liu, P. Gong, chem. Comm., 56 (2020) 519) -.

Bright fluorescence: according to our previous experimental study, the covalent organic framework material designed by us has obvious fluorescence characteristics, the fluorescence color usually appears in different solvents is slightly different, but usually shows light blue obvious fluorescence, and the fluorescence characteristics can change when we load drugs, thereby realizing the visualization of the load (see p. Gong, f. Wang, f. Guo, j. mater. chem. B, 6 (2018) 7926-7935).

Specificity of glucose oxidase: the glucose oxidase as a novel biological enzyme medicament has obvious inhibition effect on cancer cells, and simultaneously can not interfere the operation of normal cells and bring excessive physical burden to patients. (see H, Cheng, X.Y. Jiang, R.R. Zheng, Biomaterials, 195 (2019) 75-85; J, Li, Y.Li, Y.Wang, Nano Lett., 17 (2017) 6983-6990.) therefore, a novel Nano anti-cancer drug carrier is developed to be applied to load biological enzyme drugs, the delivery of the glucose oxidase to cancer cells is further improved, and the treatment effect of the glucose oxidase to cancer is improved, and the novel Nano anti-cancer drug carrier has good practical significance in both practical application and scientific research.

However, since glucose oxidase is difficult to deliver to cancer cells, and considering that the covalent organic framework material has a certain porosity and conjugation, there are no published reports and technical studies for using the covalent organic framework material to load biological enzymes.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a covalent organic framework nano-drug carrier, and the controlled release mode and the controlled release way of glucose oxidase provided by the carrier can obviously improve the bioavailability of the drug, reduce the toxic and side effects of the drug and improve the treatment effect of the drug.

The invention also provides a preparation method of the nano-drug carrier and a glucose oxidase drug load.

The invention also provides the application of the nano-drug carrier in the preparation of anti-cancer drugs.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a covalent organic framework nano-drug carrier is prepared by reacting N, N, N ', N' -tetra (4-formylphenyl) -1,1 '-biphenyl-4, 4' -diamine monomer with 3,3',5,5' -tetramethylbenzidine monomer to obtain covalent organic framework material, and then loading drug glucose oxidase through room temperature reaction.

The preparation method of the nano-drug carrier comprises the following steps:

(1) preparation of specific covalent organic framework monomer materials: adding N, N, N ', N' -tetraphenylbenzidine and imidazole into a round-bottom flask, then adding acetonitrile, dropwise adding trifluoroacetic anhydride in a nitrogen atmosphere, refluxing the mixture until the N, N, N ', N' -tetraphenylbenzidine is completely consumed, pouring the reaction solution into water to dissolve out a yellow powder precipitate, washing the filter cake with water until the filtrate becomes colorless to obtain a product monomer precursor A, dissolving the precursor A in tetrahydrofuran, then pumping HCl gas, refluxing the reaction solution for 10-20 hours, cooling the reaction solution to room temperature, and forming an orange solid; filtering the reaction mixture and recrystallizing from diethyl ether to obtain a compound monomer B;

(2) preparation of specific covalent organic framework materials: adding the compound monomer B and 3,3',5,5' -tetramethyl benzidine into a round-bottom flask, adding tetrahydrofuran, adding 0.1-10 mL of formic acid as a catalyst, carrying out reflux reaction under an oil bath pan, cooling the solution to room temperature to form a yellow-orange mixed object, carrying out suction filtration on the mixture, and drying at 25 ℃ to obtain a covalent organic framework material solid C;

(3) loading of glucose oxidase drug: dissolving the solid C in phosphate buffered saline, then sequentially adding glucose oxidase solution, stirring at room temperature in the dark, centrifuging the resulting mixture at high speed, separating the supernatant, washing the lower solid with PBS solution, and then freeze-drying to obtain a yellowish-brown solid powder.

Preferably, in the above preparation method, in the step (1), the mass ratio of N, N' -tetraphenylbenzidine to imidazole is 1 to 3.5: 0.9-3.1, 2.0-7.0 g of N, N, N ', N' -tetraphenylbenzidine is added into 90 mL of acetonitrile, and the volume ratio of the acetonitrile to the trifluoroacetic anhydride is 0.9: 0.15-0.25;

in the step (1), 8-15g of the precursor A is dissolved in 200 mL of tetrahydrofuran, the volume ratio of hydrochloric acid to tetrahydrofuran is 2:1, and the concentration of HCl gas is 2.5 mol L^-1。

Preferably, in the above preparation method, the mass ratio of the compound monomer B to 3,3',5,5' -tetramethylbenzidine in step (2): 1: 0.75 to 1.75.

Preferably, in the preparation method, the temperature of the reflux reaction in the oil bath in the step (2) is 65-75 ℃ and the time is 4-72 hours.

Preferably, in the preparation method, the mass ratio of the solid C to the glucose oxidase in the step (3) is 10: 0.5-3, the concentration of the glucose oxidase solution is 0.1-0.5 mg/mL, and the molecular weight of the glucose oxidase is 100000-200000.

Preferably, in the preparation method, the rotation speed of the centrifugation in the step (3) is 5000-8000 r/min, and the time is 5-20 min.

In the carrier prepared by the invention, the solid C covalent organic framework material is a monolayer or multilayer laminated structure or sphere with the thickness of 100-500 nm.

The application of the nano-drug carrier prepared by the method in preparing anticancer drugs.

Advantageous effects

(1) The covalent organic framework material synthesized by the light elements is used as a carrier, so that excessive physical burden caused by introducing excessive heavy metals into the carrier in the past is avoided, meanwhile, a complex synthesis procedure is avoided in a simpler synthesis process, and large-scale production is expected to be realized.

(2) Biological enzyme materials are used as medicines, so that physical function damage caused by the medicines is avoided, and targeted damage to cancer cells can be realized. Greatly reduces the toxic and side effects of the medicine.

(3) The carrier material has obvious fluorescence, certain fluorescence intensity is reduced after the enzyme drug is loaded, the phenomenon can be obviously observed under the irradiation of an ultraviolet lamp, and the visual drug loading can be realized.

(4) Can realize the lossless loading and the controllable release of the medicine. The anticancer drug is adsorbed on the carrier through strong physical adsorption, thus avoiding the damage of covalent bonds to drug molecules, and the drug loaded by the adsorption mode can be slowly released, thus being beneficial to maintaining the drug effect for a long time.

Drawings

FIG. 1 is a fluorescence spectrum of a covalent organic backbone material (COF) in different solvents;

fig. 2 is an ir spectrum of a COF carrier material with two synthetic monomers, TPB and TMB;

FIG. 3 is a photograph of a covalent organic framework material under a thermal field Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM);

FIG. 4 is an infrared spectrum of a covalent organic backbone material (COF) with a glucose oxidase drug (GOX) before and after loading;

fig. 5 is a picture of the dispersion of COF materials in different solvents;

FIG. 6 shows the viability of HeLa cells at different concentrations of GOX, COF, GOX + COF.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

1. Preparation of specific covalent organic framework monomer materials

(1) N, N, N ', N' -tetraphenylbenzidine (5 g) and imidazole (5 g) were charged to a 250 mL two-necked round bottom flask, followed by 90 mL acetonitrile. In a nitrogen atmosphere (N)₂) Trifluoroacetic anhydride (17.5 mL) was added dropwise thereto. Refluxing the mixture untilTo complete depletion of N, N' -tetraphenylbenzidine (monitored by thin layer chromatography). The reaction solution was poured into 1L of water to dissolve a yellow powder precipitate. The filter cake was washed with water until the filtrate became colorless to give the product, monomeric precursor a. Precursor A (10 g) was dissolved in 200 mL Tetrahydrofuran (THF). HCl gas (100 mL) was then pumped in by adding 21.0 mL of concentrated HCl to 79.0 mLH₂2.5 mol L prepared in O^-1). The reaction solution was refluxed for 12 hours. The reaction solution was cooled to room temperature and an orange solid was formed. The reaction mixture was filtered and recrystallized from ether to give compound monomer b (tpb).

(2) N, N, N ', N' -tetraphenylbenzidine (5 g) and imidazole (5 g) were charged to a 250 mL two-necked round bottom flask, followed by 90 mL acetonitrile. In a nitrogen atmosphere (N)₂) Trifluoroacetic anhydride (17.5 mL) was added dropwise thereto. The mixture was refluxed until complete depletion of N, N' -tetraphenylbenzidine (monitored by thin layer chromatography). The reaction solution was poured into 1L of water to dissolve a yellow powder precipitate. The filter cake was washed with water until the filtrate became colorless to give the product, monomeric precursor a. Precursor A (15 g) was dissolved in 200 mL Tetrahydrofuran (THF). HCl gas (100 mL) was then pumped in by adding 21.0 mL of concentrated HCl to 79.0mL of H₂2.5 mol L prepared in O^-1). The reaction solution was refluxed for 12 hours. The reaction solution was cooled to room temperature and an orange solid was formed. The reaction mixture was filtered and recrystallized from ether to give compound monomer b (tpb).

(3) N, N, N ', N' -tetraphenylbenzidine (5 g) and imidazole (5 g) were charged to a 250 mL two-necked round bottom flask, followed by 90 mL acetonitrile. In a nitrogen atmosphere (N)₂) Trifluoroacetic anhydride (17.5 mL) was added dropwise thereto. The mixture was refluxed until complete depletion of N, N' -tetraphenylbenzidine (monitored by thin layer chromatography). The reaction solution was poured into 1L of water to dissolve a yellow powder precipitate. The filter cake was washed with water until the filtrate became colorless to give the product, monomeric precursor a. Precursor A (10 g) was dissolved in 200 mL Tetrahydrofuran (THF). HCl gas (100 mL, by mixing 21.0)mL concentrated HCl 79.0mL H₂2.5 mol L prepared in O^-1). The reaction solution was refluxed for 16 hours. The reaction solution was cooled to room temperature and an orange solid was formed. The reaction mixture was filtered and recrystallized from ether to give compound monomer b (tpb).

(4) N, N, N ', N' -tetraphenylbenzidine (5 g) and imidazole (5 g) were charged to a 250 mL two-necked round bottom flask, followed by 90 mL acetonitrile. In a nitrogen atmosphere (N)₂) Trifluoroacetic anhydride (17.5 mL) was added dropwise thereto. The mixture was refluxed until complete depletion of N, N' -tetraphenylbenzidine (monitored by thin layer chromatography). The reaction solution was poured into 1L of water to dissolve a yellow powder precipitate. The filter cake was washed with water until the filtrate became colorless to give the product, monomeric precursor a. Precursor A (15 g) was dissolved in 200 mL Tetrahydrofuran (THF). HCl gas (100 mL) was then pumped in by adding 21.0 mL of concentrated HCl to 79.0 mLH₂2.5 mol L prepared in O^-1). The reaction solution was refluxed for 16 hours. The reaction solution was cooled to room temperature and an orange solid was formed. The reaction mixture was filtered and recrystallized from ether to give compound monomer b (tpb).

2. Preparation of covalent organic framework materials

(1) The compound monomer B (TPB) (600 mg) and 3,3',5,5' -Tetramethylbenzidine (TMB) (440 mg) were added to a 250 mL round bottom flask, 120 mL Tetrahydrofuran (THF) was added, and several drops of formic acid were added as a catalyst, and the reaction was refluxed at 70 ℃ for 3 days under an oil bath and when the solution was cooled to room temperature, a yellow orange solid was formed at about 400 nm. As shown in FIG. 3, the thermal field scanning electron microscope and the transmission electron microscope showed that the spherical nano-carrier of 400 nm was evident. The mixture was suction filtered and dried at 25 ℃ to give solid C of covalent organic framework material. As shown in FIG. 1, ab and cd are fluorescence spectra of COF material dissolved in DMSO and ethyl acetate, respectively. As shown in the ir spectrum of fig. 2, COF was successfully synthesized and compared to the two monomers. While the dispersion of COF was observed by dispersing COF in different solvents, as shown in fig. 5, COF showed good dispersion in different solvents.

(2) The compound monomer B (TPB) (600 mg) and 3,3',5,5' -Tetramethylbenzidine (TMB) (440 mg) were added to a 250 mL round bottom flask, 120 mL Tetrahydrofuran (THF) was added, and several drops of formic acid were added as a catalyst, and the reaction was refluxed at 70 ℃ for 1 day under an oil bath and when the solution was cooled to room temperature, a yellow orange solid was formed at around 200 nm. The mixture was suction filtered and dried at 25 ℃ to give solid C of covalent organic framework material.

(3) The compound monomer B (TPB) (600 mg) and 3,3',5,5' -Tetramethylbenzidine (TMB) (440 mg) were added to a 250 mL round bottom flask, 120 mL Tetrahydrofuran (THF) was added, and several drops of formic acid were added as a catalyst, and the reaction was refluxed at 75 ℃ for 3 days under an oil bath and when the solution was cooled to room temperature, a yellow orange solid was formed at about 500 nm. The mixture was suction filtered and dried at 25 ℃ to give solid C of covalent organic framework material.

(4) The compound monomer B (TPB) (600 mg) and 3,3',5,5' -Tetramethylbenzidine (TMB) (440 mg) were added to a 250 mL round bottom flask, 200 mL Tetrahydrofuran (THF) was added, and several drops of formic acid were added as a catalyst, and the reaction was refluxed at 65 ℃ for 3 days under an oil bath and when the solution was cooled to room temperature, a yellow orange solid was formed at about 500 nm. The mixture was suction filtered and dried at 25 ℃ to give solid C of covalent organic framework material.

The covalent organic framework material is a monolayer or multilayer laminated structure or sphere with the thickness of 100-500 nm.

3. Loading of glucose oxidase drug

(1) 5 mg of solid C was dissolved in 5 mL of Phosphate Buffered Saline (PBS), and then 1 mL of a glucose oxidase solution (0.5 mg/mL) having a molecular weight of 150000 was sequentially added thereto, and stirred at room temperature in the dark for 24 hours. The resulting mixture was centrifuged at high speed, the supernatant was separated, and the lower solid was washed with a PBS solution and then freeze-dried to obtain a yellowish brown solid powder. As shown in the ir spectrum of fig. 3, GOX was successfully loaded onto COF carriers.

(2) 5 mg of solid C was dissolved in 5 mL of Phosphate Buffered Saline (PBS), and then 1 mL of a glucose oxidase solution (0.1 mg/mL) having a molecular weight of 180000 was sequentially added, and stirred at room temperature in the dark for 24 hours. The resulting mixture was centrifuged at high speed, the supernatant was separated, and the lower solid was washed with a PBS solution and then freeze-dried to obtain a yellowish brown solid powder.

(3) 5 mg of solid C was dissolved in 5 mL of Phosphate Buffered Saline (PBS), and then 1 mL of a glucose oxidase solution (0.3 mg/mL) having a molecular weight of 150000 was sequentially added thereto, and stirred at room temperature in the dark for 24 hours. The resulting mixture was centrifuged at high speed, the supernatant was separated, and the lower solid was washed with a PBS solution and then freeze-dried to obtain a yellowish brown solid powder.

(4) 10 mg of solid C was dissolved in 5 mL of Phosphate Buffered Saline (PBS), and then 1 mL of a glucose oxidase solution (0.3 mg/mL) having a molecular weight of 180000 was sequentially added, and stirred at room temperature in the dark for 24 hours. The resulting mixture was centrifuged at high speed, the supernatant was separated, and the lower solid was washed with a PBS solution and then freeze-dried to obtain a yellowish brown solid powder.

4. And (3) toxicological experiments: COF cytotoxicity and COF + GOX and anticancer Activity

The carrier obtained in example 2 was mixed with different proportions of GOX to complete drug loading to obtain COF + GOX, then the unbound GOX was removed by ultrafiltration, the precipitate was redissolved with PBS and stored in a refrigerator at 4 ℃ for further use.

The HeLa cells are respectively inoculated in a 96-well plate, the GOX and the COF + GOX are respectively added after 24 hours to form a concentration gradient, and the culture medium is added for culture. Under the same condition, pure COF, pure GOX and COF + GOX loaded with GOX with the same concentration are used as parallel control, and cell survival rate detection is carried out after 24 hours.

As a result, as shown in fig. 6, it can be seen that the survival rate of HeLa cells was increased at a different concentration of GOX from COF vector. Also, experiments show that COF is not substantially toxic to cells even at high concentrations. The GOX under the same concentration is loaded by the COF carrier, so that the medicine has more excellent treatment effect and good slow-release effect.

Claims

1. A covalent organic framework nano-drug carrier is characterized in that N, N, N ', N' -tetra (4-formylphenyl) -1,1 '-biphenyl-4, 4' -diamine monomer and 3,3',5,5' -tetramethyl benzidine monomer react to obtain covalent organic framework material, and then drug glucose oxidase is loaded through room temperature reaction.

2. A method for preparing the covalent organic framework nano-drug carrier of claim 1, comprising the steps of:

(2) preparation of specific covalent organic framework materials: adding the compound monomer B and 3,3',5,5' -tetramethyl benzidine into a round-bottom flask, adding tetrahydrofuran, adding a proper amount of formic acid as a catalyst, carrying out reflux reaction under an oil bath kettle, cooling the solution to room temperature to form a yellow-orange mixture, carrying out suction filtration on the mixture, and drying at 25 ℃ to obtain a covalent organic framework material solid C;

3. The production method according to claim 2,

in the step (1), the mass ratio of the N, N, N ', N' -tetraphenylbenzidine to the imidazole is 1-3.5: 0.9-3.1, 2.0-7.0 g of N, N, N ', N' -tetraphenylbenzidine is added into 90 mL of acetonitrile, and the volume ratio of the acetonitrile to the trifluoroacetic anhydride is 0.9: 0.15-0.25;

in the step (1), 8-15g of the precursor A is dissolved in 200 mL of tetrahydrofuran, the volume ratio of hydrochloric acid to tetrahydrofuran is 2:1, and the concentration of HCl gas is 2.5 mol L^-1；

The amount of formic acid added in the step (2) is 0.1-10 mL.

4. The method according to claim 2, wherein the mass ratio of the compound monomer B to 3,3',5,5' -tetramethylbenzidine in the step (2): 1: 0.75 to 1.75.

5. The method according to claim 2, wherein the reflux reaction in the oil bath in the step (2) is carried out at a temperature of 65 to 75 ℃ for 4 to 72 hours.

6. The method according to claim 2, wherein the mass ratio of the solid C to the glucose oxidase in step (3) is 10: 0.5-3, the concentration of the glucose oxidase solution is 0.1-0.5 mg/mL, and the molecular weight of the glucose oxidase in step (3) is 100000-200000.

7. The method according to claim 2, wherein the rotation speed of the centrifugation in the step (3) is 5000-8000 r/min for 5-20 min.

8. The preparation method according to claim 2, wherein the covalent organic framework material prepared in step (2) is in a layer shape or a spherical shape with a wavelength of 100-500 nm.

9. Use of the nano-covalent organic-framework nano-drug carrier of claim 1 in the preparation of anti-cancer drugs.