Graphene modification method

Abstract

The graphene modification method comprises the following steps: step 1, preparing a reagent; step 2, synthesis of TPG; step 3, cell culture; 4, carrying out a biocompatibility experiment on the TPG; step 5, constructing a nano-drug based on TPG and DOX; step 6, characterizing the TPG and TD nano-drugs; and step 7, releasing DOX in the TD nano-drug in vitro. The TPG has good biocompatibility, has the effect of promoting cell proliferation in a certain concentration range, has large loading capacity, has the maximum theoretical loading capacity of 8.299 * 10 < 4 > mg / g for DOX, is an excellent substrate material, and is expected to be applied to the field of biomedicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical technology field, and particularly to a modification method of graphene. BACKGROUND

[0002] With the multi-disciplinary cross-fusion and rapid development of chemistry, biomedical, materials science and nanotechnology, nanomedicine has made great progress in the field of disease diagnosis and treatment. Among them, the selection of nanocarriers plays an important role. Graphene-based materials have been used as a carrier material in the field of biological medicine due to their large specific surface area, easy modification and other properties. In addition, green functionalized reduced graphene oxide not only reduces the aggregation of graphene oxide itself, but also endows it with the dual excellent properties of functional molecules and graphene, further improving the properties of the carrier material and enhancing the effect. In recent years, green tea has become one of the most popular healthy drinks in modern society, rich in tea polyphenols (TP), which has good effects on improving hypertension, hyperlipidemia, protecting heart function, and reducing the incidence of cancer. High-concentration tea can effectively improve the clinical symptoms of diabetes, improve the function of the spleen and stomach, remove metabolic waste, and reduce blood viscosity. At the same time, TP is also a good antioxidant and can be used as an excellent green reducing agent. In previous studies, TP was used to synthesize tea polyphenol functionalized reduced graphene oxide (TPG) in one step, and it was applied to the loading of heavy metal ions, BSA, cytochrome c, etc.; the modification of tissue engineering materials to promote the proliferation of osteoblasts and accelerate bone healing; and the modification of titanium implants to promote bone growth due to its excellent electrical properties. In view of its excellent biological application value, it is expected to be used as a carrier material in the field of nanomedicine. SUMMARY

[0003] In order to solve the problems existing in the prior art, the present application provides a modification method of graphene.

[0004] The technical scheme adopted by the present application to solve its technical problems is: A modification method of graphene, comprising the following steps: Step 1 reagent preparation: Step 2 synthesis of TPG: Step 3 cell culture: Resuscitate mouse fibroblast strain in culture bottle using 10% FBS and 1% penicillin streptomycin RPMI1640 medium, place it in a 37℃ incubator containing 5% CO2 for overnight adhesion growth, and after liquid change treatment, observe that the cell growth state is good and basically covers the bottom of the bottle, and then prepare for use; Step 4 TPG biocompatibility experiment: (1) Stability in various solution systems; 80ug / mL of TPG was dispersed in water, PBS (pH7.4), RPMI and RPMI+FBS medium, and the stability of TPG in different systems was observed after 6 months; (2) Toxicity to normal cells; L929 cells were cultured in 96-well plates at a density of 2x104 / well overnight, and different concentrations of TPG were added to the wells respectively, and cultured at 37℃ for 24, 48 and 72h; finally, the TPG-treated cells were washed with PBS, and CCK-8 reagent was used according to the requirements, and the determination was carried out by using the enzyme marker at 450nm excitation; Step 5: Construction of TPG and DOX nanomedicine: Step 6: Characterization of TPG and TD nanomedicine: The cytotoxicity of TPG was quantitatively analyzed by an enzyme marker; the morphology of TPG and TD was characterized by field emission scanning electron microscopy; the structure and functional group of TD were characterized by X-ray diffractometer and Fourier transform infrared spectrometer respectively; the loading capacity of TPG, drug release and fluorescence properties of nanomedicine were tested by a molecular fluorescence instrument; Step 7: In vitro release of DOX in TD nanomedicine.

[0005] The present application also has the following additional technical features: As further specific optimization of the technical scheme of the present application: the reagents are graphene oxide solution, green tea polyphenol, concentrated nitric acid, sodium hydroxide, phosphate buffer powder, adriamycin hydrochloride, experimental water is twice distilled water, fetal bovine serum and phosphate buffer solution, RPMI1640 culture medium, penicillin streptomycin and trypsin, CCK-8 kit is purchased, and mouse fibroblasts.

[0006] As further specific optimization of the technical scheme of the present application: the synthesis method of TPG is as follows: 60mg of TP powder is dissolved in 20mL of distilled water; then the TP solution is added to 20mL of 0.5mg / mL GO solution under stirring, and a uniform mixed solution is obtained after ultrasonic treatment for 10min; the above mixed solution is transferred to a reaction kettle, and reacted at 80℃ for 8h; after natural cooling, the solution is centrifuged and washed several times at 11000r / min; finally, 10kDa dialysis bag is used for dialysis to remove unreacted TP, and TPG solution is obtained.

[0007] As a further specific optimization of the technical solution of the present invention: a construction method based on TPG and DOX nanodrugs: 1mL DOX solution and 1mL 0.1mg / mL TPG were stirred and reacted at pH = 7.4 for 24h; then, centrifuged at 13000r / min for 10min, washed several times with PBS, and observed until the supernatant was basically colorless to obtain TPG-DOX nanodrugs for use; in order to explore the maximum adsorption capacity of TPG, different concentrations of DOX were reacted with TPG according to the above method, the supernatant after centrifugation was collected, and the absorbance of DOX was detected using a molecular fluorescence instrument to determine the loading efficiency of DOX on TPG. E (%) and load capacity q (mg / g), calculated as follows: E =( W 0– W 1) / W 0×100%(1); q =( W 0– W 1) / W TPG (2) where: W 0 and W 1 represents the mass of DOX added and the mass of DOX remaining in the supernatant after multiple centrifugation, mg; W TPG represents the mass of TPG added, mg.

[0008] As a further specific optimization of the technical solution of the present invention: In vitro release of DOX from TD nanomedicine: 4 mL of TD nanomedicine was placed in a 3500Da dialysis bag, and then placed in 4 mL of PBS solution with pH values ​​of 7.4, 6.8, and 5.0, respectively. After a certain period of time, 2 mL of the supernatant was taken out, and 2 mL of fresh PBS solution was added to continue soaking, and the above steps were repeated until the set time point; finally, the absorbance of DOX in the supernatant was measured using a molecular fluorescence instrument; based on the cumulative release law of the drug, the release efficiency of DOX in the TD nanomedicine within the specified time was calculated, as shown below: E =[ CnV 0+( C 1+ C 2+…+ Cn -1) V ] / W loaded×100%(3): Cn For the n The concentration of DOX in the supernatant taken out for the first time, mg / mL; V 0 is the volume of release medium, mL; V is the volume of each sample in mL; W loaded is the mass of DOX adsorbed on the material before release, mg.

[0009] Compared with the prior art, the present application has the advantages of: TPG has good biocompatibility, promotes cell proliferation within a certain concentration range, has a large loading capacity, and has a maximum theoretical loading capacity of DOX of up to 8.299*104mg / g, which is an excellent base material and is expected to be applied in the biomedical field. Through multi-method verification, TPG and DOX obtain nano drug TD through physical adsorption, TD has pH-dependent slow-release performance, and is a pH-responsive fluorescent probe. The results can lay a preliminary research foundation for the selection of drug carriers and drug monitoring in later cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 Schematic diagram of the stability of TPG in water, PBS, RPMI1640 and RPMI1640+FBS systems before and after 6 months; Figure 2 Schematic diagram of cytotoxicity analysis of different concentrations of TPG on L929 cells; Figure 3 Schematic diagram of the effect of the initial concentration of DOX on TPG loading DOX; Figure 4 Schematic diagram of Langmuir and Freundlich two adsorption isotherm models; Figure 5 Schematic diagram of the effect of pH on TD nano drug release and fluorescence recovery; Figure 6 SEM diagram of DOX, TPG and TD; Figure 7 XRD and FTIR diagrams of DOX, TPG and TD. DETAILED DESCRIPTION

[0011] The exemplary embodiments disclosed herein will be described in greater detail below with reference to the accompanying drawings. EMBODIMENT

[0012] A modification method of graphene, comprising the following steps: Step 1: Reagent preparation: Graphene oxide solution (GO) was provided by Shanxi Energy College, green tea polyphenols (TP), concentrated nitric acid (HNO3), sodium hydroxide (NaOH), phosphate buffer powder (PBS, pH = 7.4) were purchased from Chengdu Kelong Reagent Co., Ltd., doxorubicin hydrochloride (DOX) was purchased from Shanghai Bioengineering Co., Ltd., and experimental water was twice distilled water. Fetal bovine serum (FBS) and phosphate buffer solution (PBS, 0.01 mol / L, pH = 7.4) were purchased from Germany PAN Company, RPMI1640 medium, penicillin streptomycin and trypsin were purchased from Hyclone Company, and CCK-8 kit was purchased from MedChemExpress Company. Mouse fibroblast cells (L929) were provided by the State Key Laboratory of Oral Diseases of West China Hospital of Stomatology, Sichuan University.

[0013] Step 2 synthesis of TPG: TPG was synthesized according to the previous synthesis method of the research group, and the specific steps are as follows: 60 mg of TP powder was dissolved in 20 mL of distilled water. Then the TP solution was added to 20 mL of 0.5 mg / mL GO solution under stirring, and a uniform mixed solution was obtained after ultrasonic treatment for 10 min. The above mixed solution was transferred to a reaction kettle and reacted at 80°C for 8 h. After natural cooling, the solution was centrifuged and washed several times at 11000 r / min. Finally, 10 kDa dialysis bag was used to remove unreacted TP and obtain TPG solution.

[0014] Step 3 cell culture: The mouse fibroblast cell line (L929) was resuscitated in the culture bottle using 10% FBS and 1% penicillin streptomycin RPMI1640 medium, and was placed in a 37°C incubator containing 5% CO2 for adherent growth overnight. After liquid change treatment, the growth state of the cells was observed to be good and basically covered the bottom of the bottle, and was ready for use.

[0015] Step 4 biocompatibility experiment of TPG: The biocompatibility of the material was investigated from the following two aspects: (1) Stability in various solution systems. 80 μg / mL TPG was dispersed in water, PBS (pH 7.4), RPMI and RPMI+FBS medium, and the stability of TPG in different systems was observed after 6 months.

[0016] (2) Toxicity to normal cells. L929 cells were cultured in 96-well plates at a density of 2 x 104 / well overnight, and different concentrations of TPG (5, 10, 25, 50, 100, 150 and 250 pg / mL) were added to the wells, respectively, and incubated at 37°C for 24, 48 and 72 h. Finally, the TPG-treated cells were washed with PBS, and CCK-8 reagent was added as required, and the determination was performed using a microplate reader at an excitation of 450 nm.

[0017] Step 5: Construction of TPG and DOX nanodrugs: 1 mL of DOX solution was reacted with 1 mL of 0.1 mg / mL TPG at pH = 7.4 for 24 h. Then, centrifugation was performed at 13000 r / min for 10 min, and washing with PBS was performed several times until the supernatant was basically colorless, to obtain TPG-DOX (TD) nanodrugs for standby. In order to explore the maximum adsorption capacity of TPG, different concentrations of DOX (25, 50, 100, 150, 200, 250, 400 and 500 mg / L) were reacted with TPG according to the above method, and the supernatant after centrifugation was collected, and the absorbance of DOX was detected using a molecular fluorescence instrument, to determine the loading efficiency of DOX on TPG E (%), and loading capacity q (mg / g), and the calculation formula was as follows: E ( W 0– W 1) / W 0 x 100% (1). q ( W 0– W 1) / W TPG (2) wherein: W 0 and W 1 represent the mass of added DOX and the mass of residual DOX in the supernatant after multiple centrifugations, respectively, mg. W TPG represents the mass of added TPG, mg.

[0018] Step 6: Characterization of TPG and TD nanodrugs: Cytotoxicity of TPG was quantitatively analyzed by a microplate reader (VariOskan Flash 3001, Thermo, USA). The morphology of TPG and TD was characterized by field emission scanning electron microscopy (FE-SEM, Inspect F, FEI, USA). The structure and functional group of TD were characterized by X-ray diffraction (XRD, X’Pert Pro, Philips, Netherlands) and Fourier transform infrared spectrometer (FTIR, Nicolet IS10, Thermo, USA), respectively. The loading capacity of TPG, drug release and fluorescence properties of nanodrug were tested by a molecular fluorescence instrument (RF-5301PC, Shimadzu, Japan).

[0019] Step 7 In vitro release of DOX in TD nanodrug: 4 mL of TD nanodrug was placed in a dialysis bag with a molecular weight of 3500 Da, and then placed in 4 mL of PBS solution with pH of 7.4, 6.8 and 5.0, respectively. After a certain period of time, 2 mL of supernatant was taken out, 2 mL of fresh PBS solution was added for continuous soaking, and the above steps were repeated until the set time point. Finally, the absorbance of DOX in the supernatant was measured by a molecular fluorescence instrument. According to the cumulative release rule of the drug, the release efficiency of DOX in TD nanodrug within a specified time was calculated and expressed as follows: E =[ CnV 0+( C 1+ C 2+…+ Cn -1) V ] / W loaded×100%(3)wherein: Cn is the concentration of DOX in the supernatant taken out for the nth time, mg / mL. n 0is the volume of the release medium, mL. V is the volume of each sample, mL. V loadedis the mass of DOX adsorbed on the material before release, mg. W Example

[0020] Results and discussion of Example 1 Evaluation of biocompatibility of TPG: The biocompatibility of TPG was evaluated from two aspects of stability and cytotoxicity. On the one hand, the stability, 100 µg / mL of TPG was mixed with double distilled water, PBS, RPMI1640, RPMI1640+FBS at a volume ratio of 1:1, respectively, and then photographed after uniform mixing. After standing for 3 months, the dispersion stability of TPG in various solutions was observed. For example, Figure 1 ​As shown in the figure, after TPG was mixed with the four solutions and allowed to stand for 20 minutes, it showed a uniform and stable solution shape. After standing for 6 months, TPG still maintained good dispersion stability in these four systems, indicating that TPG has good stability. On the other hand, the conventional cell L929 was selected for cytotoxicity evaluation. Figure 2 As shown, when the concentration of TPG.

[0021] At concentrations up to 50 μg / mL, TPG exhibited varying degrees of proliferation effects on L929 cells, demonstrating its absolute safety and modest proliferative effects within this range. At concentrations of 100 μg / mL and above, cell viability remained elevated above 90%, but with little proliferative effect. When the concentration was further increased to 250 μg / mL, the viability of L929 cells remained above 60% after 72 hours of incubation, exceeding the IC50 limit for safety assessment, demonstrating that TPG remains cell-friendly at concentrations up to 250 μg / mL. Furthermore, at concentrations below 150 μg / mL, cell viability remained above 100%, indicating that TPG promoted cell proliferation and demonstrating that TPG exhibits proliferative effects within a certain concentration range. These findings suggest that TPG exhibits excellent biocompatibility and potential applications in biomedicine.

[0022] Effect of initial DOX concentration on TD nanodrug loading: Effect of initial DOX concentration on loading efficiency and capacity Figure 3 As shown in the figure, as the DOX concentration increases from 5 mg / L to 500 mg / L, the loading efficiency of TPG first increases and then decreases, while the loading capacity shows a gradual upward trend. When the TPG concentration is not higher than 250 mg / L, the loading efficiency increases with the increase of DOX concentration. When it is higher than 250 mg / L, the loading efficiency decreases linearly. The above phenomenon may be due to the fact that when the DOX concentration reaches 250 mg / L, the adsorption sites of TPG are almost occupied, and the excess DOX cannot be accommodated by a certain amount of TPG, resulting in a reduced loading efficiency. In terms of adsorption capacity, when the DOX concentration is not higher than 250 mg / L, TPG has sufficient adsorption sites, and the adsorption capacity increases rapidly with the increase of DOX concentration. When the DOX concentration is higher than 250 mg / L, the adsorption sites on TPG are insufficient, and a small amount of DOX continues to be loaded, and the adsorption capacity shows a slow increase trend.

[0023] Adsorption isotherm: This study used two commonly used adsorption isotherm models, Langmuir and Freundlich, to investigate the loading behavior and capacity of TPG-loaded DOX. The Langmuir model describes the loading behavior of the material as a uniform monolayer, with equal adsorption capacity at each loading site and no force between the loading and the loaded material, as shown below: (4).

[0024] The Freundlich model is an empirical equation that represents the load between the load and the loaded object in a multi-layered and uneven manner, as shown below: (5); among which: q e and C e represents the loading capacity and concentration of DOX at equilibrium, respectively; K L represents the adsorption free energy constant, q m represents the maximum load capacity; K F and 1 / n are Freundlich constants, representing adsorption constant and adsorption intensity respectively. Figure 4 As shown in Table 1, the loading of DOX by TPG conforms to the two isotherm models described above, indicating that TPG loading of DOX is a mixed single- and multi-layer loading behavior. This is likely due to the fact that TPG is in a non-uniform single- and multi-layered structure during loading, with DOX being loaded within the non-uniform lamellar structure of TPG. Furthermore, Langmuir adsorption isotherm calculations indicate that TPG has a high adsorption capacity, with a theoretical maximum adsorption capacity of 8.299 × 10⁴ mg / g, indicating that TPG's lamellar structure imparts excellent loading behavior.

[0025] Table 1 Calculation results of adsorption isotherms of TPG loaded DOX Langmuir Freundlich m (mg / g) L (10"4L / mg) 2 F (mg / g)2 8.299×104 3.568 0.9998 1.048 36.160.9878 Effect of pH on TD nanoparticle drug release: The TD nanomedicine was placed in the release system with pH values ​​of 5.0, 6.8, and 7.4, respectively. The average value was obtained after three repeated experiments, and the release efficiency was calculated according to formula (3). Among them, the pH value of the release system is 7.4, pH 6.8, and pH 5.0, which represent the normal physiological conditions, the survival conditions of cancer tissue, and the environmental conditions inside cancer cells, respectively. Figure 5(a) shows that TD nanomedicine has the highest release efficiency under acidic conditions, especially at pH 5.0, indicating that the addition of TPG effectively reduces the release of DOX in normal body fluids, thereby reducing the side effects of the drug on the normal body. At pH 5.0, 6.8 and 7.4, the DOX release efficiency is 31.36%, 11.28%, 8.57% respectively, indicating that the release of TD nanomedicine is pH dependent. At the same time, with the extension of time, the drug release efficiency increases until 80h, which shows that TD nanomedicine has a certain sustained release effect. In addition, TD nanomedicine has fluorescence switching performance, such as Figure 5 (b) shows that the addition of TPG makes the fluorescence of DOX quenched (off), and when placed in different pH PBS solutions, it shows different degrees of fluorescence recovery (on), which is a pH-dependent fluorescence switch probe, which is conducive to the imaging monitoring of drug release in later in vitro and in vivo experiments.

[0026] In summary, TD is a pH-dependent sustained-release nanomedicine, and TD has better release efficiency under pH 5.0 conditions and better dispersion stability, which helps TD nanomedicine to better circulate in the blood to reach the cancer cells after entering the body, and effectively open the fluorescence switch to release DOX, thereby monitoring the drug and chemotherapeutic treatment of cancer.

[0027] Characterization of TD nanomedicine: The morphology of TD nanomedicine before and after synthesis was observed by SEM. As shown in Figure 6 , DOX is a nanoparticle with a diameter of about 30 nm (see Figure 6 (a)), and TPG shows a wrinkled structure of lamella (see Figure 6 (b)), when TPG is used as a carrier and combined with DOX (see Figure 6 (c)), it shows a certain aggregation phenomenon, but DOX can be clearly observed to be wrapped by the lamellar structure of TPG, indicating that DOX is successfully loaded on TPG to form TD nanomedicine.

[0028] In addition, the structural changes of TD nanomedicine before and after synthesis were characterized by XRD. As shown in Figure 7 (a), pure TPG has a characteristic peak at 2 θ θ of 25.1°, indicating the successful reduction of TPG. DOX has a characteristic peak at 25.6°, and when loaded with TPG, the characteristic diffraction peak (24.2°) of TD nanomedicine has a small blue shift compared with the characteristic diffraction peak of pure TPG, indicating that the interlayer distance of TD nanomedicine increases, and DOX supports the distance between TPG lamella, further verifying that DOX is successfully wrapped and loaded by TPG. At the same time, the functional group changes of TD nanomedicine before and after synthesis were characterized by FTIR. As shown inFigure 7 (b) As shown in the FTIR spectra of TPG-DOX TD nanodrugs, the characteristic peaks of TPG at 3410 cm-1 (O—H), 1724 cm-1 (C==O in carboxyl functional group) and 1052 cm-1 (C—O in epoxy group) can be clearly observed. Meanwhile, two characteristic peaks of DOX at 1729 and 1265 cm-1 (dotted line) appeared in the FTIR spectra of TPG-DOX TD nanodrugs, indicating that TPG successfully loaded DOX. In addition, no new peaks were observed in the FTIR spectra of TD, which indirectly indicated that the loading of TPG and DOX belonged to simple physical adsorption, and no new chemical bonds were formed.

[0029] The foregoing detailed description of the embodiments of the application provided in the accompanying drawings is not intended to limit the scope of the application as claimed, but merely represents selected embodiments of the application.

Claims

1. A method for modifying graphene, characterized in that: The following steps are involved: Step 1 Reagent preparation: Step 2 Synthesis of TPG: Step 3 Cell culture: Resuscitate mouse fibroblasts in RPMI1640 medium containing 10% FBS and 1% penicillin-streptomycin in a culture flask. Place the flask in a 37°C incubator containing 5% CO2 to allow the cells to adhere to the flask overnight. After changing the medium, observe that the cells are growing well and have basically covered the bottom of the flask before use. Step 4: Biocompatibility test of TPG: (1) Stability in various solution systems: 80 μg / mL TPG was dispersed in water, PBS (pH 7.4), RPMI, and RPMI+FBS culture media, and the stability of TPG in different systems was observed after 6 months; (2) Toxicity to normal cells: L929 cells were cultured in a 96-well plate at a density of 2×104 / well overnight, and different concentrations of TPG were added to the wells and cultured at 37°C for 24, 48, and 72 hours. Finally, the TPG-treated cells were washed with PBS and measured using a microplate reader at 450 nm excitation using the CCK-8 reagent as required. Step 5: Construction of nanomedicine based on TPG and DOX: Step 6 Characterization of TPG and TD nanomedicines: The cytotoxicity of TPG was quantitatively analyzed using a microplate reader; the morphology of TPG and TD was characterized using a field emission scanning electron microscope; the structure and functional groups of TD were characterized using an X-ray diffractometer and a Fourier transform infrared spectrometer, respectively; the loading capacity, drug release, and fluorescence properties of TPG nanomedicines were tested using a molecular fluorescence instrument; Step 7 In vitro release of DOX from TD nanomedicine.

2. A method for modifying graphene according to claim 1, characterized in that: The reagents include graphene oxide solution, green tea polyphenols, concentrated nitric acid, sodium hydroxide, phosphate buffer powder, doxorubicin hydrochloride, the experimental water is double distilled water, fetal bovine serum and phosphate buffer solution, RPMI1640 culture medium, penicillin streptomycin and trypsin, CCK-8 kit purchased, mouse fibroblasts.

3. A method for modifying graphene according to claim 1, characterized in that: The TPG synthesis method is as follows: 60 mg of TP powder is weighed and dissolved in 20 mL of distilled water; the TP solution is then added to 20 mL of 0.5 mg / mL GO solution under stirring conditions, and ultrasonicated for 10 minutes to obtain a uniform mixed solution; the above mixed solution is transferred to a reactor and reacted at 80°C for 8 hours; after natural cooling, the solution is centrifuged and washed several times at 11,000 rpm; finally, dialyzed using a 10 kDa dialysis bag to remove unreacted TP and obtain a TPG solution.

4. A method for modifying graphene according to claim 1, characterized in that: The construction method of nanomedicine based on TPG and DOX is as follows: 1mL DOX solution and 1mL 0.1mg / mL TPG were stirred and reacted at pH = 7.4 for 24h; then, centrifuged at 13000r / min for 10min, washed with PBS several times, and observed until the supernatant was basically colorless to obtain TPG-DOX nanomedicine for use; in order to explore the maximum adsorption capacity of TPG, different concentrations of DOX were reacted with TPG according to the above method, and the supernatant after centrifugation was collected. The absorbance of DOX was detected using a molecular fluorescence instrument to determine the loading efficiency of DOX on TPG. E (%) and load capacity q (mg / g), calculated as follows: E =( W 0– W 1) / W 0×100%(1); q =( W 0– W 1) / W TPG (2) where: W 0 and W 1 represents the mass of DOX added and the mass of DOX remaining in the supernatant after multiple centrifugation, mg; W TPG represents the mass of TPG added, mg.

5. The method for modifying graphene according to claim 1, wherein: In vitro release of DOX from TD nanomedicine: 4 mL of TD nanomedicine was placed in a 3500 Da dialysis bag and then placed in 4 mL of PBS solutions with pH values ​​of 7.4, 6.8, and 5.0, respectively. After a certain period of time, 2 mL of the supernatant was removed and 2 mL of fresh PBS solution was added to continue soaking. This process was repeated until the set time point. Finally, the absorbance of DOX in each supernatant was measured using a molecular fluorescence instrument. Based on the cumulative release pattern of the drug, the release efficiency of DOX from the TD nanomedicine within the specified time was calculated as follows: E =[ CnV 0+( C 1+ C 2+…+ Cn -1) V ] / W loaded×100%(3): Cn For the n The concentration of DOX in the supernatant taken out for the first time, mg / mL; V 0 is the volume of release medium, mL; V is the volume of each sample in mL; W loaded is the mass of DOX adsorbed on the material before release, mg.

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