A covalent organic framework material integrating diagnosis and treatment, and its preparation method and application

Abstract

The present invention discloses a diagnosis and treatment integrated covalent organic framework material and its preparation method and application, and relates to the field of diagnosis and treatment integrated technology. The present invention uses 2,4,6-tris (4-aminophenyl) 1,3,5-triazine and 4,4'-((2,5-bis (2-ethylhexyl) 3,6-dioxo 2,3,5,6-tetrahydropyrrolo [3,4-c] pyrrole 1,4-diyl) bis (thiophene 5,2-diyl)) dibenzaldehyde as raw materials to prepare COF materials with imaging and photothermal properties, the preparation method is conventional, the reaction operation is simple and easy to control, and the yield of the prepared product is high. The nanoparticles prepared by nanoprecipitation method not only have good water solubility and biocompatibility, but also have good photothermal conversion ability, can realize imaging of tumor cells and efficient killing of various types of tumor cells, and provide new strategies for the realization of tumor diagnosis and treatment integration.

Description

Technical Field

[0001] The present invention relates to the technical field of integrated diagnosis and treatment, and in particular to a covalent organic framework material for integrated diagnosis and treatment, and a preparation method and application thereof. Background Art

[0002] As a disease that seriously affects human health and quality of life, cancer has become a major challenge in the field of public health. Accurate diagnosis and efficient treatment of tumors are of great significance to improving the treatment effect of tumors. Separating diagnosis and treatment will lead to low diagnosis and treatment efficiency, and the lack of real-time monitoring and feedback mechanism during the treatment process will affect the treatment effect. The long examination and diagnosis process often causes patients to miss the best treatment time window, which may cause patients to develop from mild symptoms to severe symptoms or even death.

[0003] By integrating cancer diagnosis and treatment functions through nanotechnology, the integrated diagnosis and treatment platform can achieve early diagnosis, precise positioning, real-time treatment monitoring and prognosis monitoring of tumors, which is conducive to achieving the best treatment effect and reducing toxic side effects. Fluorescence imaging (FLI), as a new type of non-invasive imaging method, has the advantages of non-invasiveness, high sensitivity and specificity, and large field of view real-time imaging, and is widely used in the field of integrated diagnosis and treatment. The development of therapeutic agents with fluorescence imaging capabilities will help achieve integrated diagnosis and treatment.

[0004] Traditional tumor treatment methods mainly include surgical resection, chemotherapy and radiotherapy. However, these methods have their limitations, such as being often accompanied by significant toxic side effects and being very prone to drug resistance. Photothermal therapy (PTT) is a new treatment method that converts light energy into heat energy under near-infrared light, which can kill cancer cells locally in a targeted manner. It has the advantages of being minimally invasive, long-lasting, and not prone to drug resistance, and has great development potential in the treatment of malignant tumors. By adjusting the molecular structure of photothermal materials, it can be achieved that they have both photothermal and fluorescent capabilities.

[0005] Organic materials have attracted widespread attention due to their good biocompatibility and adjustable energy bands. Among them, covalent organic frameworks (COFs) are framework materials connected by covalent bonds, which have properties such as periodicity, crystallinity, porosity, and stability. Due to its large conjugated plane, its energy band gap is relatively small, and it has potential for development in the field of photothermal therapy. In addition, its large conjugated platform also contributes to the generation of fluorescent imaging. However, there are still relatively few studies on photothermal materials based on COF; in addition, due to the large conjugated structure of COF, its water solubility is usually poor; the influence of the structure and performance of COF has greatly limited its research and application in the biological field.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a diagnosis and treatment integrated covalent organic framework material and its preparation method and application, so as to solve the above technical problems.

[0008] The present invention is achieved in that:

[0009] In the first aspect, an embodiment of the present invention provides a diagnosis and treatment integrated covalent organic framework material, the structural formula of which is shown below, wherein R is selected from -C6H 13 , -C2H5, -C4H9 and -C8H 17 Any of the following:

[0010]

[0011] In a second aspect, an embodiment of the present invention provides a method for preparing a covalent organic framework material as described above, comprising synthesizing the covalent organic framework material according to the following synthesis route:

[0012]

[0013] In a third aspect, an embodiment of the present invention provides a diagnosis and treatment integrated covalent organic framework nanoparticle, which includes the covalent organic framework material as described above; the average particle size of the covalent organic framework nanoparticle is less than 200 nm.

[0014] In a fourth aspect, an embodiment of the present invention provides a method for preparing the covalent organic framework nanoparticles as described above, the preparation method comprising the following steps:

[0015] The third compound, the fifth solvent and the amphiphilic copolymer are mixed to prepare covalent organic framework nanoparticles; wherein the fifth solvent includes DMSO; the amphiphilic copolymer includes a DSPE-PEG copolymer; further, the mass ratio of the third compound to the amphiphilic copolymer is 1:(2-4).

[0016] In a fifth aspect, an embodiment of the present invention provides an application of the covalent organic framework nanoparticles as described above in the preparation of anti-tumor drugs, wherein the tumor is selected from at least one of liver cancer, lung cancer, glioma, lymphoma, breast cancer, melanoma, colorectal cancer, cervical cancer, and pancreatic cancer.

[0017] In a sixth aspect, an embodiment of the present invention provides a use of the aforementioned covalent organic framework nanoparticles in tumor imaging.

[0018] The present invention has the following beneficial effects:

[0019] The diagnosis and treatment integrated covalent organic framework material provided by the present invention uses the first compound and the second compound as raw materials to obtain a new type of covalent organic framework material with diagnosis and treatment integrated function through Schiff base reaction. The structure is clear, the preparation method is conventional, the reaction conditions are mild, the reaction operation is simple and easy to control, and the yield is high. The prepared COF NPs are a new type of covalent organic framework nanoparticles with diagnosis and treatment integrated function. The ultraviolet absorption of the covalent organic framework nanoparticles is in the near-infrared region, and it has excellent photothermal conversion ability, which can realize the imaging and efficient killing of tumor cells, and is expected to be applied to the anti-tumor field, providing a new strategy for the realization of integrated tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 are FTIR spectra of the first compound, the second compound and the third compound;

[0022] Figure 2 The hydrated particle size distribution and potential diagram of covalent organic framework nanoparticles;

[0023] Figure 3 This is the UV-visible-near-infrared absorption spectrum of covalent organic framework nanoparticles in aqueous solution;

[0024] Figure 4 Thermal imaging pictures of aqueous solution of covalent organic framework nanoparticles under laser irradiation: (a) temperature change curve; (b) thermal imaging picture;

[0025] Figure 5 This is a temperature change curve of the aqueous solution of covalent organic framework nanoparticles under laser irradiation with different power densities;

[0026] Figure 6 This is a photo-stability test diagram of covalent organic framework nanoparticles;

[0027] Figure 7 Detection of photothermal conversion efficiency of covalent organic framework nanoparticles: (a) heating and cooling curve; (b) linear relationship diagram of -ln(θ) over time;

[0028] Figure 8The cytotoxicity results of covalent organic framework nanoparticles on different cancer cells: (a) 4T1 cells; (b) B16-F10 cells; (c) CT26 cells; (d) Panc02 cells; (e) A549 cells; (f) Hela cells;

[0029] Fig. 9 This is the fluorescence imaging of covalent organic framework nanoparticles on 4T1 cells. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0031] In the first aspect, an embodiment of the present invention provides a covalent organic framework material integrating diagnosis and treatment, the structural formula of which is shown below, wherein R is selected from -C6H 13 , -C2H5, -C4H9 and -C8H 17 Any of the following:

[0032]

[0033] In a second aspect, an embodiment of the present invention provides a method for preparing a covalent organic framework material as described above, comprising synthesizing the covalent organic framework material according to the following synthesis route:

[0034]

[0035] It should be noted that the preparation method of the covalent organic framework material in the present invention uses the first compound and the second compound as raw materials to obtain a new type of covalent organic framework material with integrated diagnosis and treatment functions through a Schiff base reaction. The material has a clear structure, a conventional preparation method, mild reaction conditions, simple and easy-to-control reaction operation, and a high yield.

[0036] In an optional embodiment of the present invention, the first compound and the second compound are dissolved respectively and then reacted with the first solvent to obtain a third compound, i.e., a covalent organic framework material (COF); wherein the first compound is 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, and the second compound is 4,4'-((2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-5,2-diyl))dibenzaldehyde, and the corresponding structural formulas are as follows: The structural formula of the first compound is The structural formula of the second compound is

[0037] The molar ratio of the first compound to the second compound is 2:(2.5-3.5); the molar ratio is selected because the molecular weights of the two compounds are quite different. In order to ensure that the reaction proceeds more thoroughly, the amount of one of the compounds needs to be increased according to the actual situation.

[0038] Furthermore, the molar ratio of the first compound to the second compound is 2:(2.9-3.2). Specifically, the molar ratio of the first compound to the second compound can be selected from any one of 2:2.9, 2:3.0, 2:3.1 and 2:3.2, or other values ​​between 2:(2.9-3.2) as needed.

[0039] In an optional embodiment of the present invention, the two compounds are first dissolved separately and then mixed with the first solvent for reaction.

[0040] Specifically, the first compound is dissolved in the second solvent by stirring at room temperature to prepare a solution, the second compound is dissolved in the third solvent by stirring at room temperature to prepare a solution, and then the two solutions are mixed to react.

[0041] Wherein, the first solvent includes an acidic substance; further, the acidic substance is an organic acid, and further a carboxylic acid; in a preferred embodiment of the present invention, the acidic substance used is glacial acetic acid.

[0042] It should be noted that the purpose of the acidic substance used in the present invention is to provide an acidic environment to promote the reaction.

[0043] In an optional embodiment of the present invention, the ratio of the volume of the acidic substance to the total volume of the second solvent and the third solvent is 1:(9-15); further 1:(10-14).

[0044] Specifically, the ratio of the volume of the acidic substance to the sum of the volumes of the second solvent and the third solvent is set to enable the first compound and the second compound to have a faster reaction efficiency and a higher conversion rate, which can be reasonably adjusted according to the actual amount of the first compound and the second compound used. In an embodiment of the present invention, the volume ratio is selected from any one of 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:12, 1:12.5, 1:13, 1:13.5 and 1:14, or other values ​​within the range of 1:(10-14).

[0045] In a preferred embodiment of the present invention, the second solvent is an alcohol solution, further methanol; and the third solvent is chloroform or dichloromethane.

[0046] In an optional embodiment of the present invention, the volume ratio of the second solvent to the third solvent is 1:(0.8-1.2), further 1:(0.9-1.1); the second solvent and the third solvent of this ratio, after being blended with the first solvent, can ensure the full dissolution of the first compound and the second compound. Specifically, the setting of the volume ratio can be reasonably adjusted according to actual conditions, and is selected from any one of 1:0.9, 1:1.0 and 1:1.1, or other values ​​within the range of 1:(0.9-1.1).

[0047] In an optional embodiment of the present invention, the method for preparing the covalent organic framework material further comprises purifying the third compound, and the purification comprises washing the third compound with a fourth solvent.

[0048] Specifically, the preparation process of the third compound is as follows: after the first compound and the second compound are dissolved respectively, they are mixed with the first solvent and allowed to stand for sedimentation, and the standing time can be reasonably adjusted according to the actual amount of the reactants; then, the solution system after sedimentation is centrifuged, the supernatant is discarded, and the precipitate is centrifuged and washed with a fourth solvent until the supernatant is colorless, and the final precipitate is placed in a vacuum drying oven for drying to finally obtain a dark purple solid COF.

[0049] The time and speed of the centrifugal treatment are reasonably adjusted according to the instrument used and the amount of solution treated. In the embodiment of the present invention, the speed is set to 8000r / min and the time is set to 10min-15min. The COF drying method can be changed and adjusted according to actual needs.

[0050] In an optional embodiment of the present invention, the fourth solvent is selected from any one of tetrahydrofuran, methanol, acetonitrile and acetone; the fourth solvent is mainly used to clean COF, therefore, COF is insoluble in the fourth solvent, and the second solvent and the third solvent are miscible with the fourth solvent. In addition, the fourth solvent is also required to have the characteristics of low boiling point and easy vacuum removal.

[0051] Furthermore, the fourth solvent is tetrahydrofuran.

[0052] It should be noted that the preparation method of the covalent organic framework material is conventional, the reaction conditions are mild, the reaction operation is simple and easy to control; the COF material obtained has a clear structure and a high yield. The ultraviolet absorption of the covalent organic framework nanoparticles is in the near-infrared region, and has excellent photothermal conversion capabilities, which can achieve efficient killing and imaging of tumor cells and can be applied to the field of integrated diagnosis and treatment.

[0053] In a third aspect, an embodiment of the present invention provides a novel covalent organic framework nanoparticle (COF NPs), which includes the covalent organic framework material as described above; the average particle size of the covalent organic framework nanoparticles is less than 200 nm.

[0054] Particles within a specific size range can achieve passive targeting of tumors and improve anti-tumor ability. The water solubility of materials can be improved during the preparation of nanoparticles, thereby increasing their potential for biological applications. In addition, the surface of nanoparticles is easy to modify to achieve the purpose of active targeted distribution. This property enables nanoparticles to be designed into materials with specific functions for use in drug delivery, bioimaging and other fields.

[0055] In addition, the COF NPs prepared by the present invention is a new type of covalent organic framework nanoparticles with integrated diagnosis and treatment functions, which effectively overcomes the problem of poor water solubility of COF due to its large conjugated structure, expands the application field of COF, and provides a new strategy for its research and development and application in the biological field.

[0056] In a fourth aspect, an embodiment of the present invention provides a method for preparing the covalent organic framework nanoparticles as described above, the preparation method comprising the following steps:

[0057] The third compound, the fifth solvent and the amphiphilic copolymer are mixed to prepare covalent organic framework nanoparticles; wherein the fifth solvent includes DMSO; and the amphiphilic copolymer is selected from any one of DSPE-PEG copolymers and PLGA-PEG copolymers.

[0058] It should be noted that COF and the amphiphilic copolymer have a large solubility in the fifth solvent, and the fifth solvent is used only as a cosolvent and does not chemically react with the solute. In addition, the fifth solvent also needs to be easily removed, and a solvent miscible with water is the best choice. Therefore, the fifth solvent in the embodiment of the present invention is DMSO. In other embodiments of the present invention, the fifth solvent can select other reasonable solvents according to actual needs.

[0059] Amphiphilic copolymers are usually composed of hydrophilic segments and lipophilic segments. This structural characteristic allows the lipophilic chains to combine with the hydrophobic structure of the material when they interact with organic materials, and expose the hydrophilic chains to water to form nanoparticles, which are widely used in imaging, sensing, and drug delivery.

[0060] In an optional embodiment of the present invention, an amphiphilic copolymer is applied to the preparation of nanoparticles; the amphiphilic copolymer used is a DSPE-PEG copolymer, which is a type of material that combines phospholipids, polyethylene glycol (PEG) and functional groups; due to the presence of PEG, DSPE-PEG has good water solubility, is easy to use in vivo, and has no toxic side effects on the organism. In addition, the introduction of PEG not only reduces the immunogenicity of the complex, but also prolongs its circulation time in the body, which helps to improve the anti-tumor effect.

[0061] Furthermore, in an optional embodiment of the present invention, the DSPE-PEG copolymer used is specifically a DSPE-PEG2000 copolymer; in other embodiments of the present invention, other specifications of DSPE-PEG copolymers or PLGA-PEG copolymers can be selected according to actual conditions.

[0062] Further, the mass ratio of the third compound to the amphiphilic copolymer is 1:(2-4), specifically selected from any one of 1:2, 1:2.5, 1:3, 1:3.5 and 1:4, or other values ​​within the range of 1:(2-4).

[0063] It should be noted that the amount of amphiphilic copolymer used in the preparation of nanoparticles is a key factor, because it directly affects the formation, stability and biocompatibility of nanoparticles. If the amount is too large, the prepared nanoparticles will have a smaller particle size, and cannot be effectively separated, resulting in unclean cleaning, which will further affect the performance of the nanoparticles; if the amount is too small, the particle size of the nanoparticles will be too large, which does not meet the requirements of the nanoscale particle size, and the utilization rate will be reduced.

[0064] In a fifth aspect, an embodiment of the present invention provides an application of the aforementioned covalent organic framework nanoparticles in anti-tumor applications.

[0065] In an optional embodiment of the present invention, different tumors are selected to study the universal applicability of the nanoparticles, and the tumor types include at least one of liver cancer, lung cancer, glioma, lymphoma, breast cancer, melanoma, colorectal cancer, cervical cancer, and pancreatic cancer. In other embodiments of the present invention, other types of tumors can be selected for research according to actual conditions.

[0066] In a sixth aspect, an embodiment of the present invention provides a use of the aforementioned covalent organic framework nanoparticles in tumor imaging.

[0067] The imaging characteristics of fluorescence imaging in the medical field mainly include high sensitivity, high resolution and real-time dynamic observation. Fluorescence imaging technology can accurately visualize tumors, which not only helps to diagnose tumors at an early stage, but also facilitates subsequent surgical treatment, improves the accuracy of surgery and the quality of life of patients, and has now been widely used in the field of integrated diagnosis and treatment.

[0068] The COF NPs prepared in the embodiment of the present invention have excellent fluorescence imaging ability, and the luminescence position thereof is mainly in the near-infrared region, with a high signal-to-noise ratio and better imaging effect, and have great application potential in tumor imaging.

[0069] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0070] Example 1

[0071] This embodiment provides a diagnosis and treatment integrated covalent organic framework material (COF), the preparation method of which comprises the following steps:

[0072] 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (first compound, 48.26 mg, 0.136 mmol) was dissolved in 150 mL of the second solvent (methanol), and 4,4'-((2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-5,2-diyl))benzaldehyde (second compound, 150 mg, 0.204 mmol) was dissolved in 150 mL of the third solvent (chloroform). The methanol solution of the first compound was stirred at room temperature, and then the chloroform solution of the second compound was added dropwise to the above solution to obtain a first mixed solution.

[0073] 30 mL of acidic substance (glacial acetic acid) was added to the first mixed solution, stirred for 1 min, and then allowed to settle for 48 h. After the reaction was completed, the mixture was centrifuged (8000 r / min, 15 min) and the supernatant was discarded.

[0074] The precipitate is purified with a fourth solvent (tetrahydrofuran), specifically by centrifugation, washed until the supernatant is colorless, and the precipitate is placed in a vacuum drying oven for drying.

[0075] Finally, a dark purple solid, the third compound, namely a covalent organic framework material (COF), was obtained, 180 mg, with a yield of 90.7%.

[0076] Example 2

[0077] This embodiment provides a diagnosis and treatment integrated covalent organic framework material (COF), which is different from Embodiment 1 in that:

[0078] 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (first compound, 60.25 mg, 0.17 mmol) was dissolved in the second solvent (methanol), and 4,4'-((2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-5,2-diyl))benzaldehyde (second compound, 200 mg, 0.272 mmol) was dissolved in the third solvent (chloroform).

[0079] Finally, a dark purple solid was obtained, namely the third compound: covalent organic framework material (COF), 230 mg, with a yield of 88.4%.

[0080] Example 3

[0081] This embodiment provides a diagnosis and treatment integrated covalent organic framework nanoparticles (COF NPs), the preparation method of which comprises the following steps:

[0082] The COF material (third compound, 2 mg) prepared in Example 1 was dissolved in 2 mL of the fifth solvent (DMSO) to prepare a second mixed solution; DSPE-PEG2000 (fourth compound, 4 mg) was dissolved in 1 mL of the fifth solvent (DMSO) to prepare a third mixed solution.

[0083] The second mixed solution and the third mixed solution were mixed and added into water under ultrasonic state, and after ultrasonic treatment for 30 minutes, the excess DMSO was removed by ultrafiltration, and finally covalent organic framework nanoparticles (COF NPs) were prepared.

[0084] Test Example 1

[0085] In order to verify the successful preparation of the covalent organic framework material (COF), the first compound, the second compound and the COF were subjected to infrared spectroscopy measurements respectively;

[0086] The specific detection steps are as follows: according to the requirements of infrared test sample preparation, the first compound, the second compound and the COF prepared in Example 1 are mixed with potassium bromide powder, ground, pressed into tablets, and tested; the specific infrared absorption spectrum is shown in Figure 1 .

[0087] from Figure 1 It can be seen that in COF, the NH absorption peak and the C=O absorption peak disappeared, and the C=N peak appeared, proving the successful preparation of COF.

[0088] Test Example 2

[0089] In this test example, the COF NPs prepared in Example 3 were subjected to hydrated particle size test and potential test. The specific test method is as follows:

[0090] COF NPs were added to the sample pool, and the particle size and potential of the solution were measured respectively. The specific particle size test results and potential test results are shown in Figure 2 .

[0091] from Figure 2 It can be concluded that the particle size of COF NPs is 137.82nm and the potential is -15.7mV, indicating the successful preparation of covalent organic framework nanoparticles.

[0092] Test Example 3

[0093] In this test example, the COF NPs prepared in Example 3 were subjected to UV absorption test, and the specific test method was as follows:

[0094] COF NPs were added to a cuvette and then measured on a UV-visible-near-infrared spectrophotometer. The specific UV-visible-near-infrared absorption spectrum can be found in Figure 3 .

[0095] from Figure 3 It can be seen that the UV absorption peak of COF NPs ranges from 500nm to 700nm, and the maximum absorption peak is 651nm.

[0096] Test Example 4

[0097] In this test example, the COF NPs prepared in Example 3 were tested by thermal imaging. The specific testing method is as follows:

[0098] S1. Prepare COF NPs into aqueous solutions with different concentrations, which are 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. 2 The temperature was recorded every 1 minute for 8 minutes, and the thermal imaging pictures were recorded every minute. Figure 4 a, thermal imaging Figure 4 b.

[0099] Combination Figure 4 a and Figure 4 b It can be seen that under laser irradiation, the temperature of the COF NPs aqueous solution increases with the increase of concentration. After 8 minutes of laser irradiation, the temperature of the 100 μg / mL solution can reach 54.5 °C, indicating that the photothermal performance of COF NPs is concentration-dependent.

[0100] Test Example 5

[0101] This test example detects the temperature change of COF NPs prepared in Example 3 under laser irradiation of different light intensities. The specific detection method is as follows:

[0102] S1. COF NPs were prepared into an aqueous solution with a concentration of 100 μg / mL.

[0103] S2. The COF NPs aqueous solution was irradiated with different light intensities under 660 nm laser, and the temperature was recorded every 1 min. The light intensities were 0 W / cm 2 , 0.25W / cm 2 , 0.5W / cm 2 , 0.75W / cm 2 , recorded for 8 minutes in total. For the specific temperature change curve, see Figure 5 .

[0104] from Figure 5 It can be seen that under laser irradiation with different light intensities, the temperature of the aqueous solution of COF NPs increases with the increase of light intensity, indicating that the photothermal properties of COF NPs are light intensity dependent.

[0105] Test Example 6

[0106] In this test example, the photostability of COF NPs prepared in Example 3 was tested. The specific testing method is as follows:

[0107] The COF NPs solution was irradiated with a 660 nm laser for 8 min, then the light source was turned off to allow the solution to cool to room temperature, and then irradiated again and cooled, and the irradiation was repeated 4 times. The specific temperature change curve is shown in Figure 6 .

[0108] from Figure 6 It can be seen that under laser irradiation, the temperature of the aqueous solution of COF NPs is almost the same after 4 cycles, indicating that COF NPs have good photostability.

[0109] Test Example 7

[0110] In this test example, the photothermal conversion efficiency of the COF NPs prepared in Example 3 was tested. The specific testing method is as follows:

[0111] S1. COF NPs were prepared into a 100 μg / mL aqueous solution.

[0112] S2. Use 660nm (0.75W / cm 2 ) After laser irradiation of COF NPs solution for 8 min, the light source was turned off to cool the solution to room temperature. The 8 min of illumination was selected as the cooling time 0 min. The subsequent time points were accumulated in sequence and θ was calculated according to formula (1):

[0113] θ=(T realtime -T sur ) / (T max -T sur )(1); In formula (1), T realtime is the actual temperature, T sur is the ambient temperature, T max is the maximum temperature.

[0114] Then, with -lnθ as the horizontal coordinate and time as the vertical coordinate, a straight line is obtained, whose slope is τ. According to formula (2), hs is calculated. COF NPs and hs water :

[0115] τ=(m×C D )hs(2); In formula (2), m is the mass of COF NPs, C D is the specific heat capacity of water.

[0116] Calculate Q by formula (3) Dis :

[0117] Q Dis =hs water ×△T water (3); Q in formula (3) Dis For energy.

[0118] Finally, the photothermal conversion rate of COF NPs was calculated according to formula (4):

[0119] η=(hs COF NPs ×△T COF NPs -Q Dis ) / I(1-10 -A660 )(4); where η is the photothermal conversion efficiency of COF NPs.

[0120] For specific temperature change curve, see Figure 7 a, -ln(θ) and time have a linear relationship. Figure 7 b.

[0121] Combination Figure 7 a and Figure 7 From the results in b, it can be seen that the photothermal conversion efficiency of COF NPs is 41.4%.

[0122] The specific calculation process is as follows: According to the experimentally measured T of COF NPs max is 51.7°C, the T of water max is 24.3℃, T sur is 21°C. By using equations (1) and (2), we can get τ COFNPs =188.85, calculate τ in the same way water = 161.24. According to τ = (m × CD) / hs, m = 0.5g, C D =4.2J / g, calculated hs COF NPs =0.0111, hs Water =0.0130. Dis =hs Water ×ΔT Water =0.0130×(24.3-21.2)=0.0403 J. The absorbance of COF NPs at 660 nm is 1.185, and the illumination intensity is 0.75 W / cm 2 , so η=(hs COFNPs ×ΔT COF NPs -Q Dis ) / I(1-10 -A660 )=(0.0111×(51.7-21.9)-0.0403) / (0.75×(1-10 -1.185 ))=41.4%.

[0123] Test Example 8

[0124] This test example studies the anti-tumor ability of COF NPs prepared in Example 3. Among them, 4T1 cells are mouse breast cancer cells, B16-F10 cells are mouse melanoma cells, CT26 cells are mouse colorectal cancer cells, Panc02 cells are mouse pancreatic cancer cells, A549 cells are human lung cancer cells, and Hela cells are human cervical cancer cells. The specific detection method is as follows:

[0125] S1. 4T1 cells, B16-F10 cells, CT26 cells, PAN02 cells, A549 cells, and Hela cells were cultured at 1×10 4 / well density was plated in a 96-well plate and cultured for 48h. Then, COF NPs and cells were co-cultured with different concentrations for 4h; the concentrations of COF NPs aqueous solution were 0μg / mL, 20μg / mL, 40μg / mL, 60μg / mL, and 80μg / mL, respectively.

[0126] S2. The cells were divided into a light group and a dark group. The cells in the light group were irradiated with 660nm laser for 10 minutes per well, while the cells in the dark group were not treated.

[0127] S3. After 3 h of continuous culture, MTT solution was added and the absorbance at 490 nm was measured on a microplate reader. The specific cytotoxicity results of COF NPs on different cancer cells are shown in Figure 8a- Figure 8 f, the cells it acts on are: Figure 8 a is 4T1 cells, Figure 8 b is B16-F10 cells, Figure 8 c is CT26 cells, Figure 8 d is Panc02 cells, Figure 8 e is A549 cells, Figure 8 f is HeLa cells.

[0128] from Figure 8 a- Figure 8 f It can be seen that for different tumor cell types, COF NPs showed low toxicity when not irradiated with light, indicating that they have good biocompatibility, while they showed high cell killing ability under light irradiation, reflecting excellent tumor treatment effects.

[0129] Test Example 9

[0130] In this test example, the COF NPs prepared in Example 3 were subjected to fluorescence imaging analysis on tumor cells. The specific test method is as follows:

[0131] S1. 4T1 cells were cultured at 1x10 5 The cells were plated at a density of 100 μg / well in a 6-well plate and divided into a control group and a COF NPs group for 36 h. COF NPs were then co-incubated with the cells for 12 h.

[0132] S2. Take out the 6-well plate and photograph it under a fluorescence microscope using white light and red fluorescence.

[0133] Specific fluorescence imaging images of COF NPs on 4T1 cells can be found in Fig. 9 .

[0134] Fig. 9 The results showed that COF NPs exhibited clear cell morphology under red fluorescence compared with the control group, indicating that they have good imaging capabilities.

[0135] In summary, the diagnosis and treatment integrated covalent organic framework material and its preparation method and application provided in the embodiment of the present invention use 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 4,4'-((2,5-bis(2-ethylhexyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]pyrrole-1,4-diyl)bis(thiophene-5,2-diyl))dibenzaldehyde as raw materials to prepare a COF material with diagnosis and treatment integrated function. The preparation method is conventional, the reaction operation is simple and easy to control, and the yield of the prepared product is high. After the COF material is prepared by DSPE-PEG2000 to obtain COF NPs, it has good water solubility and biocompatibility, and has good photothermal conversion ability under laser irradiation, which can achieve efficient killing of tumor cells and fluorescence imaging, providing a new strategy for the realization of integrated tumor diagnosis and treatment.

[0136] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A covalent organic framework material for integrated diagnosis and treatment, characterized in that: The structural formula of the covalent organic framework material is shown below, wherein R is selected from -C6H 13 , -C2H5, -C4H9 and -C8H 17 Any of the following:

2. A method for preparing a covalent organic framework material as claimed in claim 1, characterized in that: The method comprises synthesizing the covalent organic framework material according to the following synthesis route:

3. The method for preparing a covalent organic framework material according to claim 2, characterized in that: The first compound and the second compound are dissolved respectively, and then reacted with the first solvent to obtain a third compound, i.e., a covalent organic framework material; wherein the molar ratio of the first compound to the second compound is 2:(2.5-3.5); preferably 2:(2.9-3.2).

4. The method for preparing a covalent organic framework material according to claim 3, characterized in that: The first compound is dissolved in a second solvent, and the second compound is dissolved in a third solvent; The first solvent includes an acidic substance; preferably, the acidic substance is an organic acid, preferably a carboxylic acid, and more preferably glacial acetic acid.

5. The method for preparing a covalent organic framework material according to claim 4, characterized in that: The ratio of the volume of the acidic substance to the total volume of the second solvent and the third solvent is 1:(9-15); preferably 1:(10-14); Preferably, the second solvent is an alcohol solution, preferably methanol; and the third solvent is chloroform or dichloromethane.

6. The method for preparing a covalent organic framework material according to claim 2, characterized in that: The method for preparing the covalent organic framework material further comprises purifying the third compound, wherein the purification comprises washing the third compound with a fourth solvent; Preferably, the fourth solvent is selected from any one of tetrahydrofuran, methanol, acetonitrile and acetone.

7. A covalent organic framework nanoparticle for integrated diagnosis and treatment, characterized in that: It includes the covalent organic framework material as claimed in claim 1; The average particle size of the covalent organic framework nanoparticles is less than 200 nm.

8. A method for preparing covalent organic framework nanoparticles as claimed in claim 7, characterized in that: The preparation method of the covalent organic framework nanoparticles comprises the following steps: mixing the third compound, the fifth solvent and the amphiphilic copolymer to prepare covalent organic framework nanoparticles; Wherein, the fifth solvent comprises DMSO; the amphiphilic copolymer is selected from any one of DSPE-PEG and PLGA-PEG copolymers; Preferably, the mass ratio of the third compound to the amphiphilic copolymer is 1:(2-4).

9. Use of the covalent organic framework nanoparticles according to claim 7 in the preparation of anti-tumor drugs, characterized in that: The tumor is selected from at least one of liver cancer, lung cancer, glioma, lymphoma, breast cancer, melanoma, colorectal cancer, cervical cancer, and pancreatic cancer.

10. Use of the covalent organic framework nanoparticles according to claim 7 in tumor imaging.

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