NO / 5-fluorouracil double-delivery system based on Zn-MOF and application of NO / 5-fluorouracil double-delivery system
By loading 5-FU and NO onto IRMOF-3 nanomaterials, IRMOF-3-NONO@5-FU was prepared, which solved the problems of poor water solubility of 5-FU and short half-life of NO, achieving pH-responsive release and synergistic anti-tumor effects, enhancing the efficacy of chemotherapy and reducing side effects.
Patent Information
- Application Number
- CN202510698038.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-10-17
AI Technical Summary
Existing chemotherapy drugs such as 5-FU have poor water solubility and significant toxic side effects. NO has a short half-life in the body, making it difficult to precisely control its concentration and duration of action at the tumor site, resulting in poor chemotherapy efficacy and drug resistance problems.
IRMOF-3-NONO@5-FU was prepared by loading 5-FU and NO onto IRMOF-3 nanomaterials and then using physical adsorption and covalent bonding. The release of the drug was controlled by pH response, thus achieving synergistic treatment with NO and 5-FU.
It achieved high drug loading and pH-responsive release of 5-FU, enhanced the killing effect on tumor cells, showed synergistic anti-tumor effect, and reduced drug side effects.
Smart Images
Figure CN120789289A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the technical field of biomedical materials, in particular to a NO / 5-fluorouracil dual delivery system based on Zn-MOF and application thereof. BACKGROUND
[0002] Cancer is one of the major diseases threatening human health worldwide, causing millions of deaths each year. Traditional cancer treatments such as surgery, radiotherapy and chemotherapy have achieved certain effects in clinical practice, but still face many challenges. Chemotherapy is one of the important means of cancer treatment, but chemotherapy drugs often have serious side effects and drug resistance problems, resulting in poor treatment effect. Therefore, it is of great significance to develop new drug delivery systems and treatment strategies to improve the efficacy of chemotherapy drugs and reduce their side effects.
[0003] Nitric oxide (NO) as an important biological signal molecule has important research value in the field of biomedicine. Studies have found that NO plays a key role in regulating tumor angiogenesis, inducing cancer cell apoptosis and enhancing immune response in the body. However, NO cannot be directly applied in cancer treatment because its half-life in the body is extremely short, usually only a few seconds to a minute, and its chemical properties are extremely active, easily metabolized and transformed. How to accurately control the effective concentration and action time of NO at the tumor site has become a key problem in the development of NO medical applications.
[0004] Metal-organic frameworks (MOFs) as a new type of nanomaterial have advantages such as high porosity, large specific surface area and controllable structure, and show great potential in gas adsorption and storage and drug delivery. In recent years, as an anti-tumor drug delivery carrier, MOFs have been reported many times. IRMOF-3, as an important member of the Zn-MOF family, has the advantages of good stability and high biological affinity.
[0005] Fluorouracil (5-FU) as a classic chemotherapy drug is widely used in the treatment of various tumors. However, its poor water solubility, high toxicity and other shortcomings greatly limit the efficacy of 5-FU and affect the quality of life of patients. To solve this problem, 5-FU is often used in combination with other drugs, and the combined treatment method not only helps to reduce the drug resistance of tumor cells, but also reduces the amount of 5-FU used and reduces its side effects. However, there are few reports on the combination of NO therapy and 5-FU. SUMMARY
[0006] The application aims to provide a new NO / 5-fluorouracil dual delivery system with controlled release characteristics. The application adopts the following technical scheme (attached Figure 1 ):
[0007] (1) IRMOF-3 can be prepared by the method disclosed in the prior art: zinc nitrate hexahydrate and 2-amino-1, 4-benzenedicarboxylic acid are dissolved in N, N-dimethylformamide (DMF) solvent, stirred at room temperature until full dissolution. Then triethylamine is slowly added to the above solution, stirred for a certain time, the product is filtered and washed with 30 mL of dichloromethane three times, dried in a vacuum oven at 120 DEG C for 12h, to obtain a zinc-based metal organic framework material IRMOF-3.
[0008] (2) IRMOF-3 is further dispersed into a methanol solution of 5-fluorouracil and stirred for a certain time, 5-FU is loaded in IRMOF-3 by adsorption to obtain a zinc-based metal organic framework material IRMOF-3@5-FU loaded with 5-FU.
[0009] (3) IRMOF-3@5-FU powder is degassed by blowing nitrogen several times, then filled with NO under high pressure, to finally obtain a zinc-based metal organic framework material IRMOF-3-NONO@5-FU loaded with NO and 5-FU.
[0010] Further, when synthesizing IRMOF-3, the stirring speed is 600-800r / min and the stirring time is 4-6h at room temperature.
[0011] Further, when synthesizing the metal organic framework material, the molar ratio of zinc nitrate hexahydrate to 2-amino terephthalic acid is 2-3:1; the molar ratio of triethylamine to 2-amino terephthalic acid is 3:1.
[0012] Further, the mass ratio of IRMOF-3 to 5-FU is 2:1.
[0013] Further, the concentration of 5-FU in methanol is 5-6mg / mL.
[0014] Further, the pressure of NO gas is 0.3-0.4mpa and the reaction time is 2-3h.
[0015] In the prepared metal organic framework composite material IRMOF-3-NONO@5-FU, 5-FU enters the pores of IRMOF-3 by physical adsorption, and the drug loading capacity can reach 48.23wt%; NO is covalently combined with the amino sites of IRMOF-3 in the form of NONOate, and has pH responsiveness, the release amount of nitric oxide is 0.36mmol / g when pH is about 5, and the release amount of nitric oxide decreases to 0.29mmol / g when pH is about 7.4. Figure 7 IRMOF-3-NONO@5-FU is easy to obtain raw materials, simple to prepare, and the release of 5-FU has pH responsiveness. Figure 7), the drug release can be regulated by pH value; the anti-tumor experiment results show that IRMOF-3-NONO@5-FU shows stronger cytotoxicity to 4T1 cells at a concentration of 25 μg / mL compared with the control product IRMOF-3@5-FU loaded with 5-FU only, and exhibits synergistic anti-tumor effect, which has good application prospect in the clinical treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 is the preparation process diagram of the IRMOF-3-NONO@5-FU material in Example 1.
[0017] Figure 2 is the X-ray diffraction (XRD) image of the IRMOF-3, IRMOF-3@5-FU, IRMOF-3-NONO@5-FU material in Example 1.
[0018] Figure 3 is the infrared (FTIR) spectrum of the IRMOF-3, IRMOF-3@5-FU, IRMOF-3-NONO@5-FU material in Example 1.
[0019] Figure 4 is the scanning electron microscope (SEM) image of the IRMOF-3, IRMOF-3@5-FU, IRMOF-3-NONO@5-FU material in Example 1.
[0020] Figure 5 is the absorbance-concentration standard curve diagram of 5-FU in methanol.
[0021] Figure 6 is the drug release curve diagram, wherein a is the 5-FU release curve of IRMOF-3@5-FU in different pH buffers, and b is the 5-FU release curve of IRMOF-3-NONO@5-FU in different pH buffers.
[0022] Figure 7 is the cumulative release amount of NO of IRMOF-3-NONO@5-FU in different pH buffers in Example 1.
[0023] Figure 8 is the 4T1 cell toxicity diagram of IRMOF-3, IRMOF-3@5-FU, IRMOF-3-NONO@5-FU at different concentrations. DETAILED DESCRIPTION
[0024] The technical solutions in the embodiments of the present application will be described clearly and completely. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.
[0025] Example 1
[0026] (1) 1.19 g of zinc nitrate hexahydrate and 0.36 g of 2-amino-1,4-benzenedicarboxylic acid were dissolved in 30 ml of DMF solvent, and stirred at room temperature until completely dissolved. Then 0.833 ml of triethylamine was slowly added to the above solution, and stirred at room temperature at a speed of 600 r / min for 4 hours. The product was filtered and washed with 30 ml of dichloromethane three times, and dried in a vacuum oven at 120°C for 12 h, to obtain a light yellow powder sample IRMOF-3.
[0027] (2) 50 mg of 5-FU was dissolved in 10 ml of methanol, and then 100 mg of IRMOF-3 was ultrasonically dispersed in the methanol solution of 5-fluorouracil. The mixture was stirred at room temperature at a speed of 400 r / min for 72 h, and collected by centrifugation at a speed of 3000 r, and washed with dichloromethane three times, and dried in an oven at 50°C, to obtain IRMOF-3@5-FU.
[0028] (3) 1 g of IRMOF-3@5-FU powder was placed in a reaction bottle, and degassed by purging with nitrogen for several times, and then filled with 0.4 mpa of NO for reaction for 2 h. After the reaction was completed, the excess NO was removed by purging with nitrogen for 10 min, to obtain sample IRMOF-3-NONO@5-FU.
[0029] Comparative Example 1
[0030] (1) 1.8 g of zinc nitrate hexahydrate and 0.36 g of 2-amino-1,4-benzenedicarboxylic acid were dissolved in 30 ml of DMF solvent, and stirred at room temperature until completely dissolved. Then 0.833 ml of triethylamine was slowly added to the above solution, and stirred at room temperature at a speed of 600 r / min for 6 hours. The product was filtered and washed with 30 ml of dichloromethane three times, and dried in a vacuum oven at 120°C for 12 h, to obtain a light yellow powder sample IRMOF-3.
[0031] (2) 60 mg of 5-FU was dissolved in 10 ml of methanol, and then 100 mg of IRMOF-3 was ultrasonically dispersed in the methanol solution of 5-fluorouracil. The mixture was stirred at room temperature at a speed of 400 r / min for 72 h, and collected by centrifugation at a speed of 3000 r, and washed with dichloromethane three times, and dried in an oven at 50°C, to obtain IRMOF-3@5-FU.
[0032] (3) 1 g of IRMOF-3@5-FU powder was placed in a reaction bottle, and degassed by purging with nitrogen for several times, and then filled with 0.3 mpa of NO for reaction for 3 h. After the reaction was completed, the excess NO was removed by purging with nitrogen for 10 min, to obtain sample IRMOF-3-NONO@5-FU
[0033] Comparative Example 2
[0034] (1) Dissolve 1.19 g of zinc nitrate hexahydrate and 0.36 g of 2-amino-1,4-benzenedicarboxylic acid in 30 ml of DMF and stir at room temperature until completely dissolved. Then, slowly add 0.833 ml of triethylamine to the solution and stir at 800 r / min at room temperature for 6 hours. The product is filtered and washed three times with 30 mL of dichloromethane. Dry in a vacuum oven at 120°C for 12 hours to obtain a light yellow powder sample IRMOF-3.
[0035] (2) Same as Example 1.
[0036] (3) 1 g of IRMOF-3@5-FU powder was placed in a reaction bottle, purged with nitrogen several times for degassing, and then filled with 0.4 MPa of NO for 3 h. After the reaction, nitrogen was flushed for 10 minutes to remove excess NO, obtaining the sample IRMOF-3-NONO@5-FU.
[0037] Verification Example
[0038] Experimental Example 1
[0039] X-ray diffraction experiments
[0040] Experimental steps: X-ray diffraction measurements were performed on IRMOF-3, IRMOF-3@5-FU and IRMOF-3-NONO@5-FU obtained in Example 1 and the comparative example, respectively.
[0041] Experimental results:
[0042] Depend on Figure 2 It can be seen that the crystal structure of the metal-organic framework material IRMOF-3 is not affected after loading 5-FU and simultaneously loading 5-FU and NO, and still maintains a complete crystal structure. The characteristic peak of 5-FU is at 2θ=16.5° in the figure, which means that 5-FU is successfully loaded.
[0043] Experimental Example 2
[0044] Fourier transform infrared spectroscopy experiment
[0045] Experimental steps: Use Fourier infrared analyzer to perform functional group determination on IRMOF-3, IRMOF-3@5-FU and IRMOF-3-NONO@5-FU obtained in Example 1 and the comparative example, respectively.
[0046] Experimental results:
[0047] Depend on Figure 3It can be seen that compared with the infrared spectrum, IRMOF-3-NONO@5-FU is 2831cm -1 The characteristic peak of 5-fluorouracil appeared at 1040-1043 cm -1 (NO), 1310–1320cm -1 (NO) and 1493–1505 cm -1 The stretching frequency of (NN) is the characteristic absorption peak of NONOates, proving the successful loading of NO.
[0048] Experimental Example 3
[0049] Scanning electron microscopy experiments and elemental mapping experiments
[0050] Experimental steps: Scanning electron microscopy was used to perform scanning electron microscopy on IRMOF-3, IRMOF-3@5-FU, and IRMOF-3-NONO@5-FU obtained in the embodiment and the comparative example, and elemental mapping experiments were performed on IRMOF-3-NONO@5-FU.
[0051] Experimental results:
[0052] Depend on Figure 4 IRMOF-3 exhibits a regular tetrahedral morphology. After loading with 5-FU, its morphology and size remain similar to those of IRMOF-3. Further loading with nitric oxide increases surface roughness, blunting edges and the appearance of localized depressions. IRMOF-3-NONO@5-FU contains the elements C, N, F, Zn, O, and F.
[0053] Experimental Example 4
[0054] Determination of drug loading of products
[0055] Experimental steps:
[0056] The 5-FU-loaded samples were separated from the mixture using a centrifuge and washed with a minimal amount of phosphate-buffered saline (PBS) to eliminate weakly adsorbed drug molecules on the surface. The amount of drug loaded in the studied samples was measured using ultraviolet-visible (UV-Vis) spectroscopy at 266 nm, and the drug loading and encapsulation efficiency of IRMOF-3@5-FU were calculated from the fitted standard curve and the following formula.
[0057] The drug loading calculation formula is as follows:
[0058] Drug loading = (drug content in MOF / total mass of MOF) × 100%
[0059] The encapsulation efficiency is calculated as follows:
[0060] Encapsulation efficiency = (drug amount in MOF / total drug mass) x 100%
[0061] Experimental results:
[0062] From Figure 5 The drug loading of IRMOF-3-NONO@5-FU was calculated to be 45.53% and the encapsulation efficiency was 96.46% according to the standard curve fitting.
[0063] Experimental Example 5
[0064] Experimental procedure:
[0065] Drug release experiments of IRMOF-3-NONO@5-FU and IRMOF-3@5-FU of Example 1 were carried out in PBS with pH of 7.4 and 5.0, respectively. Specifically, 5 mg of the sample of Example was taken and the drug release experiment was carried out in 10 mL of PBS. At different time intervals (0.5, 1, 2, 3, 4, 6, 8, 10, 22, 35, 48, 58, 60 h), a certain volume of supernatant was taken, centrifuged to remove possible trace precipitate, then the taken volume of PBS with corresponding pH was supplemented, and the possible precipitate was moved back to the release solution. The absorbance was measured at a wavelength of 266 nm, and the drug release amount was calculated according to the standard curve of 5-fluorouracil.
[0066] Experimental results:
[0067] From Figure 6 The cumulative release curves of 5-FU of IRMOF-3@5-FU and IRMOF-3-NONO@5-FU in different pH environments were shown. Both IRMOF-3@5-FU and IRMOF-3-NONO@5-FU showed significant pH-responsive drug release characteristics. At pH = 5 (simulating the acidic environment of tumor), both of them showed an initial "burst effect", and then entered a slow release stage, in which the cumulative release rate of IRMOF-3@5-FU was nearly 100% at 60 h, while that of IRMOF-3-NONO@5-FU was about 70%; in pH = 7.4 (simulating normal physiological environment), the release rates of both were significantly reduced, and the cumulative release amount was limited. The difference in carrier structure led to different drug release kinetics, and IRMOF-3@5-FU was more sensitive to acidic environment and had higher release efficiency.
[0068] Experimental Example 6
[0069] Experimental procedure:
[0070] (1) NaNO2 standard solution was diluted with PBS buffer of pH = 7.4 and pH = 5 to obtain a series of NaNO2 standard solutions (0.625, 1.25, 2.5, 5, 10, 20 μmol / mL), then 1 ml was taken into a test tube, 0.5 mL of 1% sulfanilamide solution, 0.5 mL of 0.1% N-1- tea ethylenediamine hydrochloride solution, and 1 ml of PBS buffer of pH 7.4 or pH 5 were added to each test tube, and the mixture was incubated at room temperature for 20 min in the dark, and finally the absorbance at 540 nm was detected by UV spectrophotometer to obtain the standard curve of NaNO2 under the conditions of pH = 5 and pH = 7.4.
[0071] (2) 10 mg of sample was weighed and dispersed in 20 mL of PBS buffer (pH 5.0 and 7.4), 1 mL of reaction solution was taken out at 5, 10, 15 min, the same volume of reaction solvent was added after sampling, 0.5 mL of 1% sulfanilamide solution, 0.5 mL of 0.1% N-1- tea ethylenediamine hydrochloride solution, and 1 mL of PBS buffer (pH 5.0 and 7.4) were added to each test tube, and the mixture was incubated at room temperature for 20 min in the dark, and the absorbance at 540 nm was measured by UV spectrophotometer, and the NO release amount was calculated according to the standard curve of NaNO2.
[0072] Experimental results:
[0073] From Figure 6 The NO release performance of IRMOF-3-NONO@5-FU under different pH conditions was demonstrated. The data showed that the NO release amount of the material at pH = 5 was significantly higher than that at pH = 7.4. This confirmed that IRMOF-3-NONO@5-FU had pH-responsive NO release characteristics, and could trigger more efficient NO release in the simulated tumor micro-acidic environment (pH = 5), while the release amount was relatively low at pH = 7.4 close to the normal physiological environment.
[0074] Experimental Example 7
[0075] Cytotoxicity experiment
[0076] Experimental procedure:
[0077] Selected 4T1 cells (100 μL / well) were seeded into 96-well plates and pre-incubated in an incubator for 24 hours. The experiment was divided into five groups: a blank control group (no cells added), a negative control group (cells added but no drug added), a 5-FU group (5-FU added, positive control group), a MOF group (IRMOF-3 added), and a 5-FU-loaded MOF group (IRMOF-3@5-FU, IRMOF-3-NONO@5-FU, converted to the equivalent concentration of 5-FU). 100 μL of drug solution was added to each group, resulting in concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, respectively. After 24 hours of incubation, the culture medium in the well plate was removed, and the cells were washed twice with 200 μL of PBS buffer. The cells were then incubated with complete culture medium containing 10% CCK-8 reagent at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader to determine the percentage of remaining cell viability.
[0078] The cell survival rate formula is as follows:
[0079]
[0080] Experimental results:
[0081] Depend on Figure 7 The results showed that at concentrations of 6.25 to 50 μg / mL, IRMOF-3@5-FU maintained higher cell viability than the 5-FU group at the same concentrations; however, at concentrations of 50 to 200 μg / mL, cell viability was lower than the 5-FU group. Furthermore, cell viability in the IRMOF-3-NONO@5-FU group was consistently lower than that in the IRMOF-3@5-FU group and even lower than that in the 5-FU group at 25 μg / mL, indicating that NO in IRMOF-3-NONO@5-FU indeed synergistically enhances the anti-tumor effect of 5-FU.
Claims
1. A Zn-MOF-based NO / 5-fluorouracil dual delivery system IRMOF-3-NONO@5-FU, characterized by: The delivery system uses IRMOF-3 as a carrier and loads 5-fluorouracil by physical adsorption. NO is covalently bound to the amino site of the IRMOF-3 ligand 2-aminoterephthalic acid in the form of NONOate.
2. The IRMOF-3-NONO@5-FU according to claim 1, characterized in that The drug loading amount of 5-fluorouracil is 40-50 wt%, and the NO loading amount is 0.20-0.30 mmol / g.
3. A method for preparing IRMOF-3-NONO@5-FU according to claim 1 or 2, characterized in that Use the following steps: (1) Dissolve zinc nitrate hexahydrate and 2-amino-1,4-benzenedicarboxylic acid in N,N-dimethylformamide (DMF) solvent and stir at room temperature until completely dissolved. Then, slowly add triethylamine to the above solution and stir to react. The product is filtered and washed with dichloromethane and dried to obtain a zinc-based metal organic framework material IRMOF-3. (2) IRMOF-3 is then dispersed in a methanol solution of 5-fluorouracil and stirred to obtain a zinc-based metal organic framework material IRMOF-3@5-FU loaded with 5-fluorouracil; (3) The IRMOF-3@5-FU powder was purged with nitrogen several times for degassing, and then NO was filled under high pressure to finally obtain the zinc-based metal organic framework material IRMOF-3-NONO@5-FU loaded with NO and 5-fluorouracil.
4. The preparation method according to claim 3, characterized in that The specific steps are as follows: in the preparation of IRMOF-3, the stirring speed is 600-800 r / min, and the stirring time is 4-6 hours.
5. The preparation method according to claim 3, characterized in that The molar ratio of the zinc nitrate hexahydrate to 2-aminoterephthalic acid is 2-3:1, and the molar ratio of 2-aminoterephthalic acid to triethylamine is 1:
3.
6. The preparation method according to claim 3, characterized in that The mass ratio of IRMOF-3 to 5-fluorouracil is 2:
1.
7. The preparation method according to claim 3, characterized in that The concentration of 5-fluorouracil in methanol is 5-6 mg / mL.
8. The preparation method according to claim 3, characterized in that The NO gas pressure is 0.3-0.4 MPa; the reaction time is 2-3 hours.
9. Use of the IRMOF-3-NONO@5-FU according to claim 1 in anti-tumor treatment.