CN104524594B - Medicine of nanometer diamond surface modification load methotrexate (MTX) and preparation method thereof - Google Patents

Abstract

The invention provides a nano-diamond surface-modified drug loaded with methotrexate, a preparation method thereof, and an application of the drug in preparing antitumor drugs. In the present invention, the surface of the nano-diamond is firstly modified with a cross-linking agent polyethylene glycol diamine, which is coupled with glycidol through an amide reaction, and then the target drug ND- PEG‑GLY‑MTX. The cell cycle and apoptosis experiments tested by MTT experiment and flow cytometry show that ND‑PEG‑GLY‑MTX can induce tumor cell apoptosis and has a stronger curative effect than free MTX, which can be used in the preparation of antitumor drugs.

Description

Drug loaded with methotrexate on nanodiamond surface modification and preparation method thereof

technical field

The invention relates to a nano-medicine, in particular to a nano-medicine loaded with a chemotherapeutic drug methotrexate after nano-diamond surface modification, a preparation method thereof, and an application of the drug in the preparation of an antitumor drug.

Background technique

With the rapid development of today's science and technology, the environment in which people live is becoming increasingly harsh, leading to an increasing incidence of malignant tumors. Chemotherapy is one of the most convenient and effective methods in the current clinical traditional cancer treatment methods, but most of the anticancer drugs are small molecular compounds, which will be quickly distributed throughout the body after blood administration, causing relatively large toxic and side effects to patients. The amount of loss is large, and multiple administrations will cause tumor cells to develop multidrug resistance, thus making the chemotherapy efficiency extremely low. Therefore, it is extremely urgent to develop new anticancer drugs with high efficiency and low toxicity.

In recent years, with the organic combination of nanoscience, life science, medicine and other disciplines, nanobiomedicine has become one of the emerging interdisciplinary subjects that attract people's attention. In view of the unique advantages of nanomaterials, the research and development of nanomedicine has great development prospects in the field of medicine. Among them, nanoparticles are used as drug carriers, and drugs are bound to nanoparticles by physical adsorption or covalent coupling. for drug delivery. Due to the characteristics of the lowest toxicity, high biocompatibility and easy surface modification among all carbon nanomaterial derivatives, nanodiamonds have great potential value as drug carriers in the field of biomedicine. However, methotrexate is an anti-folate chemotherapeutic drug that is widely used in clinical practice. After blood administration, it damages normal cells in the systemic circulation process and causes drug loss at the same time. In view of the superiority of nano-diamond drug loading and drug delivery, we prepared nano-drugs by chemically coupling methotrexate to the modified nano-diamonds, and studied its effects on tumor cell viability, cycle, apoptosis, etc.

Contents of the invention

The object of the present invention is to provide a nano-diamond surface-modified drug loaded with methotrexate, a preparation method thereof, and an application of the drug in antitumor drugs.

The preparation method of the medicine that a kind of nano-diamond surface modification load-loaded methotrexate provided by the invention comprises the following steps:

(1) Weigh dry carboxylated nano-diamonds, add 1-1.5mL concentration of 0.1M, pH5.8 MES buffer solution for every 1mg of carboxylated nano-diamonds, ultrasonically disperse for 30min to form a suspension, and press Add 0.2 mg EDC and 0.3 mg NHS to 1 mg of carboxylated nano-diamonds, stir and react at room temperature for 6 hours, centrifuge at 15,000 rpm for 5 minutes after the timing is over, discard the supernatant, and obtain activated carboxylated nano-diamond precipitates;

(2) Disperse the activated carboxyl nano-diamond precipitate into a boric acid buffer solution with a concentration of 0.1M and pH 8.4, ultrasonically disperse to form a suspension, and add 0.5 mg of H ₂ N-PEG per 1 mg of activated carboxyl nano-diamond -NH ₂ , continue to stir the reaction at room temperature for 12 hours. After the reaction is completed, use a boric acid buffer solution with a concentration of 0.1M and a pH of 8.4 to wash 3 times with centrifugation at 15,000 rpm for 5 minutes to obtain nanodiamond-polyethylene glycol diamine (ND-PEG -NH ₂ ) precipitate;

(3) Dissolve every 1mg of ND-PEG- _NH2 precipitate in 0.5mL of absolute ethanol, disperse by ultrasonic for 30min to form a suspension, add drop by drop for every 1mg of ND-PEG- _NH2 precipitate with a volume fraction of 1 % glycidol ethanol solution 0.5mL, then add a few drops of triethylamine, maintain the pH of the reaction system at 8-9, stir and react at room temperature in the dark for 24h, and obtain a precipitate, which is dehydrated with absolute ethanol and sterilized double distilled water respectively. Centrifuge at 15000rpm for 5min and wash 3 times to obtain the nano-diamond-polyethylene glycol diamine-glycidol (ND-PEG-GLY) carrier, which is stored in a vacuum drying oven away from light;

(4) Add 1 mL of sterilized double-distilled water for every 1 mg of dry ND-PEG-GLY, and disperse in the dark for 30 minutes to form a suspension, then press the mass ratio of ND-PEG-GLY to methotrexate (MTX) to be 5: 1-3 Add MTX, then add 0.6mg EDC and 0.35mg NHS to each 1mg MTX, react in a 37°C water bath for 24 hours in the dark, centrifuge at 15000rpm for 5min, wash with sterilized double-distilled water for 3 times, and prepare nano-diamonds The drug ND-PEG-GLY-MTX loaded with methotrexate on the surface was vacuum-dried and refrigerated.

Compared with the prior art, the present invention has beneficial effects: the nano-diamond material selected in the present invention has many advantages such as good biocompatibility, non-toxicity, stable chemical properties, and easy surface modification. The nanocarrier ND-PEG-GLY interacted with human cervical cancer cells (HeLa), indicating that ND-PEG-GLY has biocompatibility; the nanocarrier ND-PEG-GLY was coupled with the chemotherapy drug methotrexate (MTX ), prepared into ND-PEG-GLY-MTX nano-medicine, the MTT experiment and flow cytometry test cell cycle and apoptosis experiments showed that ND-PEG-GLY-MTX can induce tumor cell apoptosis and the curative effect is stronger than free MTX, It can be used in the preparation of antitumor drugs.

Description of drawings

Figure 1A Scatter plot of HeLa cells detected by flow cytometry

Figure 1B is a scatter diagram of the uptake of nanomedicine ND-PEG-GLY-MTX by HeLa cells detected by flow cytometry

Figure 2 Effect of nanomedicine ND-PEG-GLY-MTX on the activity of HeLa cells cultured in vitro

Fig. 3A The results of HeLa cell cycle detected by flow cytometry

Fig. 3B Detecting the effect of methotrexate MTX on the HeLa cell cycle by flow cytometry

Fig. 3C Detecting the effect of nanocarrier ND-PEG-GLY on HeLa cell cycle by flow cytometry

Fig. 3D Detecting the effect of nanomedicine ND-PEG-GLY-MTX on HeLa cell cycle by flow cytometry

Figure 3E Figure 3A-D The histogram of cell cycle phase comparison

Fig. 4 Histogram of each experimental group's effect on cell apoptosis with the prolongation of treatment time

Figure 5 Mechanism of endocytic nanomedicine ND-PEG-GLY-MTX

detailed description

The following are the materials used in the examples:

Nanodiamond (ND, element six, about 140nm in diameter, Ireland).

Methotrexate (MTX) is an anti-folate chemotherapeutic drug with a molecular formula of C ₂₀ H ₂₂ N ₈ O ₅ and a molecular weight of 454.45. It is produced by Jiangsu Hengrui Medicine Co., Ltd., and its specifications are calculated based on C ₂₀ H ₂₂ N ₈ O ₅ 100mg.

Glycidol (GLY, molecular weight 74.08) was produced by Sigma Company.

Polyethylene glycol diamine (H ₂ N-PEG-NH ₂ , molecular weight 2000) was produced by Shanghai Xibao Biological Co., Ltd.

Example 1:

(1) Preparation of nano-diamond-polyethylene glycol diamine (ND-PEG) composite

Accurately weigh 5mg of carboxylated nano-diamonds, dry in a vacuum oven at 70°C overnight, place in 5mL of MES (0.1M, pH5.8) buffer solution, disperse ultrasonically for 30min to form a suspension, then add 1.0mg of EDC, 1.5 mg NHS, stirred at room temperature for 6 hours, centrifuged at 15,000 rpm for 5 minutes, removed the supernatant, and obtained nano-diamond precipitates with activated carboxyl groups;

Redisperse the nano-diamonds with activated carboxyl groups into boric acid (BBS, 0.1M, pH8.4) buffer solution, ultrasonically disperse for 30min to form a suspension, add 2.5mg of H ₂ N-PEG-NH ₂ , and continue stirring at room temperature for 12h. Wash with boric acid buffer solution (BBS, 0.1M, pH8.4) and centrifuge at 15000rpm for 5min three times to obtain nanodiamond-polyethylene glycol diamine (ND-PEG-NH ₂ ) precipitate, which is dried in vacuum for later use.

(2) Preparation of nano-diamond-polyethylene glycol diamine-glycidol (ND-PEG-GLY) nanocarrier

Accurately weigh 5mg of dry ND-PEG, then add 2.5mL of boric acid (BBS0.1M, pH8.4) buffer solution, ultrasonically disperse for 30min to form a suspension, add 2.5mL of glycidyl ethanol with a volume fraction of 1% dropwise solution, and then drop a few drops of triethylamine into the reaction solution to maintain the pH of the reaction system at about 8, stir and react at room temperature in the dark for 24 hours, centrifuge at 15,000 rpm for 5 minutes, remove the supernatant, and wash twice with sterilized double-distilled water. The supernatant was discarded to obtain the ND-PEG-GLY nanocarrier precipitate, which was dried in vacuo for later use.

(3) Preparation of nanodiamond-polyethylene glycol diamine-glycidol-methotrexate (ND-PEG-GLY-MTX) nanomedicine

Accurately weigh 5mg of dry ND-PEG-GLY, ultrasonically disperse and resuspend in 5mL sterilized double distilled water, then place it in a 37°C water bath, then quickly add 2mg MTX, 1.2mg EDC, 0.7mg NHS, avoid light The reaction was stirred for 24h. After the reaction is over, centrifuge at 15000rpm for 5min, wash the precipitate with sterilized double distilled water until the supernatant is colorless, collect all the supernatant and record its volume, to determine the unreacted MTX, the prepared nano drug ND- PEG-GLY-MTX was vacuum dried for later use.

In boric acid buffer solution (BBS) at pH 8.0, prepare MTX solution with a concentration of 2.2M, dilute and prepare 2.2×10 ^-3 M, 6.6×10 ^-3 M, 11×10 ^-3 M, 15.4×10 ^-3 M, 19.8×10 ^-3 M, 33×10 ^-3 M, 44×10 ^-3 M, 55×10 ^{-3 M} , 66×10 ^-3 M, the absorbance was measured at 303nm by UV-Vis spectrophotometry value. Take the MTX concentration as the abscissa and the absorbance value at 303 nm as the ordinate to draw the standard curve of MTX. According to the standard curve formula, the mass of MTX in the supernatant after the reaction was subtracted from the total amount of MTX added before the reaction, and the mass of MTX per mg of ND-PEG-GLY coupling was (132±1.75) μg.

Example 2:

(1)～(2) are prepared with embodiment 1

(3) Preparation of nanodiamond-polyethylene glycol diamine-glycidol-methotrexate (ND-PEG-GLY-MTX) nanomedicine

Accurately weigh 5mg of dry ND-PEG-GLY, ultrasonically disperse and resuspend in 5mL sterile double-distilled water, then place it in a 37°C water bath, then quickly add 1mg MTX, 1.2mg EDC, 0.7mg NHS, avoid light The reaction was stirred for 24h. After the reaction, centrifuge at 15,000rpm for 5min, wash the precipitate with sterilized double distilled water until the supernatant is colorless, collect all the supernatant and record its volume, and measure the absorbance of MTX at 303nm by a UV-visible spectrophotometer Based on this, the coupling MTX per mg of ND-PEG-GLY was calculated as (102 ± 0.25) μg.

Example 3:

(1)～(2) are prepared with embodiment 1

(3) Preparation of nanodiamond-polyethylene glycol diamine-glycidol-methotrexate (ND-PEG-GLY-MTX) nanomedicine

Accurately weigh 5 mg of dry ND-PEG-GLY, ultrasonically disperse and resuspend in 5 mL of sterilized double distilled water, then place it in a 37°C water bath, then quickly add 3 mg of MTX, 1.2 mg of EDC, 0.7 mg of NHS, and keep away from light The reaction was stirred for 24h. After the reaction, centrifuge at 15,000rpm for 5min, wash the precipitate with sterilized double distilled water until the supernatant is colorless, collect all the supernatant and record its volume, and measure the absorbance of MTX at 303nm by a UV-visible spectrophotometer Based on this, the coupling MTX per mg of ND-PEG-GLY was calculated to be (122±0.6) μg.

Example 4:

The Experiment of Detecting the Uptake of Nanomedicine (ND-PEG-GLY-MTX) by HeLa Cells by Flow Cytometry

HeLa cells in the logarithmic growth phase were inoculated in a 35mm culture dish at a density of 2.0×10 ⁵ /dish, cultured for 16 hours, discarded, and washed with PBS. In this experiment, the ND-PEG-GLY-MTX prepared in Example 1 was used For nanomedicine, add ND-PEG-GLY-MTX (containing 100 μg/mL MTX) and incubate at 37°C for 4 hours. After incubation, collect the cells and measure the fluorescence intensity of the cells by flow cytometry. For control.

Figure 1A and Figure 1B are scatter diagrams of forward scatter intensity (FSC) and side scatter intensity (side scatter intensity, SSC) of HeLa cells, FSC reflects the relative size of the cell, and SSC reflects the size of the cell The complexity of the particulate matter, the more particles, the greater the SSC scattering intensity. Comparing Figures 1B and 1A, it was observed that the SSC signal after the ND-PEG-GLY-MTX nanomedicine interacted with the cells was significantly stronger than the SSC intensity of the cells themselves, indicating that the particles in the cells increased, which was due to the nanomedicine ND-PEG -GLY-MTX enters into cells in large quantities, which shows that ND-PEG-GLY-MTX nanomedicine can enter into HeLa cells. However, there was no significant difference in the intensity of FSC between the two, indicating that the size of the cells basically did not change, indicating that the nano-medicine would not affect the properties of the cells themselves.

Example 5:

Effect of ND-PEG-GLY-MTX nanomedicine on HeLa cell viability

HeLa cells in the logarithmic growth phase (10% FBS/DMEM medium, 5% CO ₂ , 37°C) were inoculated into 96-well culture plates at 5×10 ³ cells per well, and after the cells adhered to the wall, replace 200 μL of 10% Each experimental group prepared in FBS/DMEM medium (A: ND-PEG-GLY 230.8 μg/mL, ND-PEG-GLY-MTX 230.8 μg/mL (including MTX 30 μg/mL), MTX 30 μg/mL; B: ND- PEG-GLY 384.6μg/mL, ND-PEG-GLY-MTX 384.6μg/mL (including MTX 50μg/mL), MTX 50μg/mL; C: ND-PEG-GLY 769.2μg/mL, ND-PEG-GLY- MTX 769.2 μg/mL (including MTX100 μg/mL), MTX 100 μg/mL), each group uses untreated HeLa cells as the blank control group, and each group has 6 replicate wells, and after culturing for 48 hours, 20 μL of 5 mg/mL MTT in PBS solution, continue to incubate for 4 hours, then discard the old culture solution in the well, add 150 μL DMSO to each well, shake evenly for 10 minutes, and perform microplate reader detection on the machine, the results are shown in Figure 2.

Figure 2 is the effect of each experimental group on the proliferation of HeLa cells. Compared with the blank cell group, it was found that the nanomaterial ND-PEG-GLY had little effect on the activity of HeLa cells under different concentration conditions, indicating that the ND-PEG-GLY carrier was biocompatible; with the increase of the drug concentration, the nanomedicine Both ND-PEG-GLY-MTX and free MTX have different degrees of killing effects on cells, and the killing effect of large concentrations of ND-PEG-GLY-MTX on cells is greater than that of free MTX, indicating that ND-PEG-PEG-MTX nanomedicine Can kill tumor cells more effectively.

Embodiment 6:

Effect of ND-PEG-GLY-MTX nanomedicine on HeLa cell cycle

In order to further study the effect of nanomedicine ND-PEG-GLY-MTX on the activity of HeLa cells, we used flow cytometry to analyze the phase of cell cycle. HeLa cells in the logarithmic growth phase were seeded in 35mm culture dishes at a density of 2.0×10 ⁵ /dish, and treated with MTX, ND-PEG-GLY, and ND-PEG-GLY-MTX after the cells adhered to the wall (each group The drug dose is the same as the above MTT experiment), after culturing for 48 hours, discard the old solution, wash with PBS for 3 times, collect the cells and fix them with 70% cold ethanol overnight, centrifuge before going to the machine, wash with PBS, and then add RNase to remove RNA for the test The interference of the results was finally stained with PI for 30 minutes, and tested on the machine, and the cells without any treatment were used as the control.

Figure 3A, B, C, and D are the PI staining results of the cells in the control group and the cells treated with MTX, ND-PEG-GLY, and ND-PEG-GLY-MTX, respectively. The figure includes a tall and thin G1 peak, a Relatively flat S-phase plateau and a short and thick G2 peak, the proportion of cells in each cell cycle can be obtained by fitting statistics on the area of each peak. It can be seen from Figure 3E that compared with the control group, the S phase of the cells in the MTX and ND-PEG-GLY-MTX treatment groups was significantly increased, and the G2 phase was significantly decreased, indicating that the drug selectively acts on the S phase, that is, the cell cycle arrest. Stagnating in S phase, blocking the process of cancer cells from S phase to G2 phase, blocking cell mitosis, hindering the synthesis of DNA and RNA, promoting cell apoptosis, inhibiting tumor cell growth, and clinically reported MTX toxicity mechanism is the same. This conclusion indicates that the nano-drug ND-PEG-GLY-MTX enters the cells and the ester bond is broken to release MTX to play an anticancer effect.

Embodiment 7:

ND-PEG-GLY-MTX Nanomedicine Induced HeLa Cell Apoptosis Experiment

Through the cell cycle effect experiment, it is known that the entry of ND-PEG-GLY-MTX nanomedicine into the cells can indeed inhibit the division and proliferation of HeLa cells, so we designed three different culture times to quantitatively detect the apoptosis of HeLa cells. Similarly, the HeLa cells in the logarithmic growth phase were seeded in a 35mm culture dish at a density of 2.0×10 ⁵ /dish, and were treated with MTX, ND-PEG-GLY, and ND-PEG-GLY-MTX after the cells adhered to the wall ( The drug dosage of each group is the same as the above-mentioned MTT experiment), and three groups were set up in parallel. After culturing for 24h, 48h, and 72h respectively, the old solution was discarded, washed 3 times with PBS, and the adherent cells were dissociated with EDTA-free trypsin and collected in fresh In the culture medium, wash with PBS before going on the machine, add Ca2 ⁺ -containing buffer solution (Binding buffer) after washing, add newly mixed double dyes (PI+Annexin-V) to it and stain for 15 minutes before going on the machine for testing.

Figure 4 shows the cell apoptosis in each experimental group with different treatment time, and the HeLa cells without any treatment were used as the control. It can be seen from the figure that with the extension of time, the apoptosis of the nanocarrier ND-PEG-GLY treatment group is not much different from that of the control group, indicating that the material has good biocompatibility; while the nanomedicine ND-PEG-GLY- Both MTX and free MTX can induce cell apoptosis, and after 72 hours, the lethality of the nanomedicine ND-PEG-GLY-MTX on cells is significantly greater than that of free MTX, which indicates that the nanomedicine prepared by us can improve the antitumor effect. Inhibition of cells.

Embodiment 8:

Mechanism of uptake of ND-PEG-GLY-MTX nanomedicine in HeLa cells detected by flow cytometry

In order to explore the transport mechanism of endocytosed ND-PEG-GLY-MTX nano-medicines, the interaction between ND-PEG-GLY-MTX nano-medicines and cells was studied with low temperature and different inhibitors. ND-PEG-GLY-MTX nanomedicine), the results are shown in Figure 5. It can be seen from the figure that when cultured at 4°C, compared with 37°C, the uptake of ND-PEG-GLY-MTX nanomedicine by HeLa cells was significantly reduced, and the inhibition rate reached 60%, which was due to the fluidity of cell membrane lipids at 4°C The interaction between exogenous substances and cell membranes is affected, so the uptake rate of exogenous substances by cells is reduced; HeLa cells are pretreated with NaN ₃ , an inhibitor that affects the energy required for endocytosis (consumes intracellular ATP) The post-inhibition rate was 2.73%, indicating that the cells take up the nano-drug almost no energy; in order to further explore the way of endocytosis of ND-PEG-GLY-MTX into the cells, the clathrin disruptor sucrose (Sucrose) and caveolin HeLa cells were pretreated with the protein disruptor methylated-β-cyclodextrin (M-β-CD), respectively. It can be seen from Figure 5 that the inhibition rate of Sucrose on the uptake of ND-PEG-GLY-MTX by HeLa cells was 16.2%, and the inhibition rate of uptake of ND-PEG-GLY-MTX by M-β-CDHeLa cells reached 75%. This infers that ND-PEG-GLY-MTX enters cells by caveolin-mediated endocytic pathway. Based on the above results, the results indicated that ND-PEG-GLY-MTX entered the cells in a temperature-dependent manner and entered HeLa cells through caveolin-mediated endocytosis.

In summary, the effect of ND-PEG-GLY-MTX on HeLa cells was tested by MTT, and the experiment of measuring cell cycle and apoptosis in different treatment groups by flow cytometry, compared with free methotrexate, fully demonstrated that The ND-PEG-GLY-MTX nanomedicine prepared by coupling the chemotherapy drug methotrexate MTX through the ester bond coupling of the nanocarrier ND-PEG-GLY can kill the tumor cells more effectively on the cancer cells. Therefore, the nanomedicine ND-PEG-GLY-MTX can be used in the preparation of highly effective antitumor drugs.

Claims (4)

1. a preparation method of a medicine for nano-diamond surface modification loaded methotrexate, characterized in that, comprising the steps: (1) Weigh dry carboxylated nano-diamonds, add 1-1.5mL concentration of 0.1M, pH5.8 MES buffer solution for every 1mg of carboxylated nano-diamonds, ultrasonically disperse for 30min to form a suspension, and press Add 0.2 mg EDC and 0.3 mg NHS to 1 mg of carboxylated nano-diamonds, stir and react at room temperature for 6 hours, centrifuge at 15,000 rpm for 5 minutes after the timing is over, discard the supernatant, and obtain activated carboxylated nano-diamond precipitates; (2) Disperse the activated carboxyl nano-diamond precipitate into a boric acid buffer solution with a concentration of 0.1M and pH 8.4, ultrasonically disperse to form a suspension, and add 0.5 mg of H ₂ N-PEG per 1 mg of activated carboxyl nano-diamond -NH ₂ , continue to stir the reaction at room temperature for 12 hours. After the reaction is completed, use a boric acid buffer solution with a concentration of 0.1M and a pH of 8.4 to wash 3 times with centrifugation at 15,000 rpm for 5 minutes to obtain nanodiamond-polyethylene glycol diamine (ND-PEG -NH ₂ ) precipitate; (3) Dissolve every 1mg of ND-PEG- _NH2 precipitate in 0.5mL of absolute ethanol, disperse by ultrasonic for 30min to form a suspension, add drop by drop for every 1mg of ND-PEG- _NH2 precipitate with a volume fraction of 1 % glycidol ethanol solution 0.5mL, then add a few drops of triethylamine, maintain the pH of the reaction system at 8-9, stir and react at room temperature in the dark for 24h, and obtain a precipitate, which is dehydrated with absolute ethanol and sterilized double distilled water respectively. Centrifuge at 15000rpm for 5min and wash 3 times to obtain the nano-diamond-polyethylene glycol diamine-glycidol (ND-PEG-GLY) carrier, which is stored in a vacuum drying oven away from light; (4) Add 1 mL of sterilized double-distilled water for every 1 mg of dry ND-PEG-GLY, and disperse in the dark for 30 minutes to form a suspension, then press the mass ratio of ND-PEG-GLY to methotrexate (MTX) to be 5: 1-3 Add MTX, then add 0.6 mg EDC and 0.35 mg NHS to each 1 mg MTX, react in a water bath at 37°C in the dark for 24 hours, centrifuge at 15,000 rpm for 5 minutes, wash with sterilized double distilled water for 3 times, and prepare nano-diamond surface modification Drug ND-PEG-GLY-MTX loaded with methotrexate, vacuum dried, and refrigerated. 2. the preparation method of the medicine of a kind of nano-diamond surface modification load methotrexate as claimed in claim 1, is characterized in that, in the described step (4), the mass ratio of ND-PEG-GLY and methotrexate It is 5:2. 3. The nano-diamond surface modification as prepared by the method as claimed in claim 1 or 2 is loaded with the medicine of methotrexate. 4. The application of the nano-diamond surface-modified drug loaded with methotrexate as claimed in claim 3 in the preparation of antitumor drugs.