A metal organic framework composite material for loading anticancer drugs using pore space partitioning method and preparation method thereof

Abstract

The present invention discloses a metal-organic framework composite material for loading anticancer drugs using a pore space partitioning (PSP) method and a preparation method thereof, and belongs to the fields of biomedicine and materials chemistry. The present invention first adopts a "one-pot method" strategy to synthesize a zeolite imidazole metal-organic framework composite material loaded with macromolecular ursolic acid, and the pore space of ZIF-8 is partitioned by ursolic acid; then a small molecule 5-fluorouracil drug is adsorbed into the remaining pore space of ZIF-8 by a physical adsorption method, and a zeolite imidazole metal-organic framework composite material loaded with two anticancer drugs is obtained. The composite material synthesized by the present invention has uniform size, and the pore space partitioning (PSP) method is used to achieve effective encapsulation of two guest molecules, making full use of the pore space of ZIF-8. The material of the present invention has a good drug pH controlled release effect, especially for small molecule 5-fluorouracil, it also has pH controlled release ability, which cannot be achieved before the pore space partitioning, and well expands the practical application field to utilize the combined action of two anticancer drugs, and then realizes the demand for treating cancer, and has certain clinical application potential.

Description

Technical Field

[0001] The invention relates to a metal organic framework composite material loaded with anticancer drugs by utilizing a pore space partitioning method and a preparation method thereof, and belongs to the fields of biomedicine and material chemistry. Background Art

[0002] Cancer seriously threatens human life and health, affects the quality of human life, and has become a major global public health problem, and its incidence is still increasing. 5-Fluorouracil (5-FU), as a thymidylate synthase inhibitor, has good efficacy and a broad anti-tumor spectrum, but is also highly toxic. It is currently a preferred drug for the treatment of solid tumors. Ursolic acid (UA) belongs to the pentacyclic triterpenoids and has multiple biological activities such as anti-cancer, anti-inflammatory, anti-diabetic, liver protection, antiviral, immunity enhancement, and prevention of cardiovascular and cerebrovascular diseases. Among them, anti-cancer activity is one of the better effects of ursolic acid among its many pharmacological activities, and it has few side effects and low toxicity, showing great potential for clinical application. Studies have shown that 5-fluorouracil and ursolic acid have a high interaction index and have a synergistic effect in treating tumors.

[0003] Metal-Organic Framework (MOFs) is a one-dimensional, two-dimensional or three-dimensional periodic porous material formed by self-assembly of organic ligands and metal ions. It has the advantages of porosity, large specific surface area, adjustable pore size and adjustable porosity, easy functionalization, catalytic activity, etc. These significant advantages make MOFs show great application potential in tumor treatment. Among them, zeolitic imidazole framework (ZIF) is a very important subtype of MOF.

[0004] At present, there have been many reports on the use of metal organic framework material ZIF-8 as a drug carrier. However, loading only one anticancer drug not only makes it difficult to fully utilize the pore space of MOFs, resulting in a waste of pore space, but also has a poor therapeutic effect on tumors when delivering a single drug. At the same time, studies have found that due to the small size of the small molecule 5-fluorouracil, it is difficult to achieve a sustained release effect when loaded in a carrier. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the utilization rate of ZIF-8 pore space is insufficient, the controlled release effect of only loading 5-fluorouracil drug is poor, and the therapeutic effect of delivering a single drug on tumors is not ideal.

[0006] In view of the deficiencies in the above-mentioned prior art, the present invention provides a zeolite imidazole metal organic framework composite material that simultaneously loads two anticancer drugs using a pore space partitioning method and a preparation method thereof, the preparation process of the method is simple, easy to operate, and the process is stable and reliable. The ZIF-8 simultaneously loads two anticancer drugs, 5-fluorouracil and ursolic acid, which can have both the efficient anticancer effect of 5-fluorouracil and the small toxic and side effects and multiple pharmacological activities of ursolic acid.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] A zeolite imidazole metal organic framework composite material that uses a pore space partitioning method to load anticancer drugs, wherein the loaded anticancer drugs are ursolic acid and 5-fluorouracil, and the molecular formula of the framework composite material is 5-FU&UA@ZIF-8. The ursolic acid in the composite material is encapsulated in the ZIF-8 pores and divides the pore space of ZIF-8, and the 5-fluorouracil is adsorbed into the pores divided by the ursolic acid.

[0009] Furthermore, in the above metal-organic framework composite material, the 5-fluorouracil loading can reach 11.2 wt%, and the ursolic acid loading can reach 12.80 wt%; the ZIF-8 is a nano-sized metal-organic framework material synthesized by a hydrothermal method with 2-methylimidazole as a ligand and zinc ions as metal ions.

[0010] The method for preparing a zeolite imidazole metal-organic framework material loaded with two anticancer drugs by using a pore space partitioning method comprises the following steps: ultrasonically dissolving zinc nitrate hexahydrate and ursolic acid in anhydrous methanol, dissolving 2-methylimidazole in a sodium hydroxide aqueous solution, and then fully mixing and stirring the two to obtain a zeolite imidazole metal-organic framework composite material loaded with ursolic acid, which can also be referred to as UA@ZIF-8, wherein the ursolic acid in the composite material is encapsulated in the ZIF-8 pores and divides the pore space of ZIF-8, and then dispersing UA@ZIF-8 into an aqueous solution of 5-fluorouracil and stirring, wherein the 5-fluorouracil is adsorbed into the pores divided by the ursolic acid, so that the sustained release of 5-fluorouracil can be achieved, and the premature leakage of small molecule drugs can be avoided; finally, a zeolite imidazole metal-organic framework composite material loaded with ursolic acid and 5-fluorouracil is obtained, which can also be referred to as 5-FU&UA@ZIF-8.

[0011] Furthermore, the ZIF-8 is a metal organic framework (MOF) material, and ZIF-8 can be prepared by methods disclosed in the prior art.

[0012] Furthermore, when synthesizing the ursolic acid-loaded zeolite imidazole metal-organic framework composite material (UA@ZIF-8), the reaction was stirred at room temperature, the stirring speed was 500~600 r / min, and the stirring time was 0.5~0.6 hours.

[0013] Furthermore, when synthesizing ursolic acid-loaded zeolite imidazole metal-organic framework composite material (UA@ZIF-8), the volume ratio of anhydrous methanol to sodium hydroxide aqueous solution was 1:1.

[0014] Furthermore, when synthesizing the ursolic acid-loaded zeolite imidazole metal-organic framework composite (UA@ZIF-8), the mass ratio of zinc nitrate hexahydrate to ursolic acid was 5:1, and the concentration of zinc nitrate hexahydrate in anhydrous methanol was 15 mg / mL.

[0015] Furthermore, when synthesizing the ursolic acid-loaded zeolite imidazole metal-organic framework composite (UA@ZIF-8), the concentration of 2-methylimidazole in the sodium hydroxide aqueous solution was 33 mg / mL, and the pH value of the sodium hydroxide aqueous solution was 12.

[0016] The zeolite imidazole metal-organic framework composite material (UA@ZIF-8) loaded with ursolic acid obtained according to the above method can continue to adsorb 5-fluorouracil by physical adsorption. UA@ZIF-8 is dispersed in an aqueous solution of 5-fluorouracil and stirred to finally obtain a zeolite imidazole metal-organic framework composite material (5-FU&UA@ZIF-8) loaded with ursolic acid and 5-fluorouracil.

[0017] Furthermore, the mass ratio of the UA@ZIF-8 and 5-fluorouracil is 5:8.

[0018] Furthermore, the concentration of 5-fluorouracil in deionized water is 5-6 mg / mL.

[0019] Furthermore, the UA@ZIF-8 is stirred in a 5-fluorouracil aqueous solution at a stirring speed of 300-500 r / min for 24 hours at room temperature.

[0020] The present invention divides the pore space of ZIF-8 by macromolecular UA, and small molecule 5-FU occupies the remaining pore space of ZIF-8 by physical adsorption, so that it has the pH-responsive co-release ability of 5-fluorouracil and ursolic acid drugs, which to a considerable extent satisfies the synergistic tumor treatment effect of the two anticancer drugs, can further enhance the anti-tumor efficacy and reduce the occurrence of drug resistance, and has a good clinical application prospect.

[0021] The present invention is based on a hydrothermal method, and adopts a "one-pot method" to first synthesize a zeolite imidazole metal-organic framework composite material (UA@ZIF-8) loaded with ursolic acid, and then utilizes the porous adsorption of the MOF material to physically adsorb the small molecule 5-fluorouracil into the voids of ZIF-8. Compared with the metal-organic framework composite material loaded with one anticancer drug, the zeolite imidazole metal-organic framework composite material loaded with two anticancer drugs synthesized by the present invention makes better use of the pores of the MOF material, and shows better anti-tumor effects and reduces the generation of drug resistance. In addition, it has been experimentally confirmed that the material exhibits a good pH-controlled release effect during the release of the two drugs, especially the small molecule 5-fluorouracil also has this feature, which greatly promotes the application progress of the synergistic effect of various types of anticancer drugs in the field of cancer tissue treatment.

[0022] The specific beneficial effects of the present invention are:

[0023] (1) The present invention adopts a one-pot synthesis followed by physical adsorption to synthesize a zeolite imidazole metal organic framework composite material loaded with two anticancer drugs, and the preparation method is simple, and the process is stable and reliable. The zeolite imidazole metal organic framework composite material loaded with two anticancer drugs adopts the pore space partitioning (PSP) method to achieve effective encapsulation of two guest molecules, which not only greatly enhances the void utilization rate of the ZIF-8 material, but also increases the overall drug loading. It has been verified that the drug loading of 5-fluorouracil is 11.2 wt%, and the drug loading of ursolic acid is 12.80 wt%.

[0024] (2) The zeolite imidazole metal organic framework composite material loaded with two anticancer drugs of the present invention exhibits a good pH controlled release effect in the release process of 5-fluorouracil and ursolic acid drugs. The macromolecular ursolic acid is effectively encapsulated in the pores because its molecular size is larger than the pore size of ZIF-8, and divides the pore space of ZIF-8. The small molecule 5-fluorouracil is adsorbed into the pores divided by ursolic acid. Due to the strong host-guest interaction caused by the spatial restriction effect, the sustained release of 5-fluorouracil can be achieved, avoiding the early leakage of small molecule drugs.

[0025] (3) The present invention utilizes zeolite imidazole metal organic framework materials to load two different types of anticancer drugs: 5-fluorouracil and ursolic acid, which play a comprehensive and synergistic anticancer effect, improve the anticancer activity of ursolic acid, reduce the toxic side effects of 5-fluorouracil, and achieve the characteristics of long-term and sustained-release anticancer effects, providing a new direction for the application of anti-tumor drugs and potential possibilities for their clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1X-ray diffraction (XRD) patterns of ZIF-8, 5-FU@ZIF-8, UA@ZIF-8, and 5-FU&UA@ZIF-8;

[0027] Figure 2 Scanning electron microscope (SEM) images and SEM element mapping images, where a is the SEM image of ZIF-8, b is the SEM image of 5-FU@ZIF-8, c is the SEM image of UA@ZIF-8, d is the SEM image of 5-FU&UA@ZIF-8, and e is the element mapping image of 5-FU&UA@ZIF-8;

[0028] Figure 3 The nitrogen adsorption isotherms of ZIF-8, 5-FU@ZIF-8, UA@ZIF-8, 5-FU&UA@ZIF-8 and the pore size distribution diagrams of ZIF-8, UA@ZIF-8, 5-FU&UA@ZIF-8;

[0029] Figure 4 This is the UV absorption spectrum of 5-FU and UA after 5-FU&UA@ZIF-8 was destroyed by hydrochloric acid and then neutralized and fixed to volume;

[0030] Figure 5 are drug release curves, where a is the principle diagram of pH-controlled drug release achieved by the pore space partitioning (PSP) method, b is the 5-FU release curve of 5-FU@ZIF-8 in different pH buffers, c is the 5-FU release curve of 5-FU&UA@ZIF-8 in different pH buffers, and d is the UA release curve of 5-FU&UA@ZIF-8 in different pH buffers;

[0031] Figure 6 The cytotoxicity of ZIF-8, 5-FU@ZIF-8, UA@ZIF-8, and 5-FU&UA@ZIF-8 to 4T1 cells at different concentrations;

[0032] Figure 7 Cell uptake diagram of 5-FU&UA@ZIF-8 co-incubated with 4T1 cells for different time periods;

[0033] Figure 8 Live and dead staining images of cells co-incubated with ZIF-8, 5-FU@ZIF-8, UA@ZIF-8, and 5-FU&UA@ZIF-8 for 4 hours. DETAILED DESCRIPTION

[0034] The contents of the present invention will be further explained below with reference to examples, but these examples do not limit the protection scope of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the protection scope of the present invention.

[0035] Example 1

[0036] (1) Using a one-pot hydrothermal method, 150 mg of zinc nitrate hexahydrate and 25 mg of ursolic acid were ultrasonically dissolved in 10 mL of anhydrous methanol, and 330 mg of the organic ligand 2-methylimidazole was dissolved in 10 mL of sodium hydroxide solution with a pH of 12, and then the two were thoroughly mixed. The reaction was stirred at 500 r / min for 0.5 h at room temperature, collected by centrifugation, washed with anhydrous methanol for 3 to 5 times, and dried in an oven at 50°C overnight to obtain 62.5 mg of ursolic acid-loaded zeolitic imidazole metal-organic framework material, referred to as UA@ZIF-8.

[0037] (2) 80 mg of 5-fluorouracil was ultrasonically uniformly dissolved in 15 mL of deionized water. Then, 50 mg of the UA@ZIF-8 prepared above was ultrasonically dispersed in the aqueous solution of 5-fluorouracil. The mixture was stirred at 400 r / min at room temperature for 24 h. The mixture was collected by centrifugation and washed with anhydrous methanol for three times. It was dried in an oven at 50°C overnight to obtain 20.6 mg of zeolitic imidazole metal-organic framework material loaded with ursolic acid and 5-fluorouracil, referred to as 5-FU&UA@ZIF-8.

[0038] Comparative Example 1

[0039] Using a one-pot hydrothermal method, 150 mg of zinc nitrate hexahydrate and 25 mg of ursolic acid were ultrasonically dissolved in 10 mL of anhydrous methanol, 330 mg of the organic ligand 2-methylimidazole was dissolved in 10 mL of sodium hydroxide solution with a pH of 12, and then the two were thoroughly mixed. The reaction was stirred at 500 r / min for 0.5 hours at room temperature, collected by centrifugation and washed with anhydrous methanol three times, and dried in an oven at 50°C overnight to obtain 62.5 mg of ursolic acid-loaded zeolite imidazole metal-organic framework material, referred to as UA@ZIF-8.

[0040] Comparative Example 2

[0041] (1) Using the solvothermal method, 400 mg of zinc nitrate hexahydrate was ultrasonically dissolved in 12 mL of anhydrous methanol, and 800 mg of the organic ligand 2-methylimidazole was dissolved in 2 mL of anhydrous methanol, and then the two were thoroughly mixed. The reaction was stirred at 400 r / min for 3 hours at room temperature, collected by centrifugation, washed with anhydrous methanol for 3 to 5 times, and dried in an oven at 50°C overnight to obtain 38.5 mg of zeolitic imidazole ester framework material, referred to as ZIF-8.

[0042] (2) 80 mg of 5-fluorouracil was ultrasonically uniformly dissolved in 15 mL of deionized water, and then 50 mg of the prepared ZIF-8 was ultrasonically dispersed in the aqueous solution of 5-fluorouracil. The reaction was stirred at 400 r / min at room temperature for 16 h, collected by centrifugation and washed with anhydrous methanol three times, and dried in an oven at 50°C overnight to obtain 15.8 mg of zeolite imidazole metal-organic framework material loaded with 5-fluorouracil, referred to as 5-FU@ZIF-8.

[0043] Verification Example

[0044] Experimental Example 1

[0045] X-ray diffraction experiments

[0046] Experimental steps: X-ray diffraction measurements were performed on ZIF-8, 5-FU@ZIF-8, UA@ZIF-8 and 5-FU&UA@ZIF-8 obtained in Example 1 and the comparative example using an X-ray diffractometer.

[0047] Experimental results:

[0048] Depend on Figure 1 It can be seen that the crystal structure of the zeolite imidazolate framework material ZIF-8 is not affected after loading 5-FU, UA, and loading 5-FU and UA at the same time. After loading the drugs, the crystal structure remains intact.

[0049] Experimental Example 2

[0050] Scanning electron microscopy experiments and elemental mapping experiments

[0051] Experimental steps: Use a scanning electron microscope to perform scanning electron microscopy on ZIF-8, 5-FU@ZIF-8, UA@ZIF-8, and 5-FU&UA@ZIF-8 obtained in the embodiment and the comparative example, and perform element mapping experiments on 5-FU&UA@ZIF-8.

[0052] Experimental results:

[0053] Depend on Figure 2 It can be seen that ZIF-8 exhibits a regular polyhedral morphology. After loading 5-FU, UA, and 5-FU and UA at the same time, the morphology and size are not significantly different from ZIF-8, and their sizes are all around 120 nm. 5-FU&UA@ZIF-8 contains C, N, F, Zn, and O elements.

[0054] Experimental Example 3

[0055] BET specific surface area test experiment

[0056] Experimental steps: The BET specific surface area and pore size distribution of ZIF-8, UA@ZIF-8, 5-FU&UA@ZIF-8 obtained in the examples and comparative examples were measured using nitrogen adsorption-desorption isotherms at 77K and vacuum conditions.

[0057] Experimental results:

[0058] Depend on Figure 3 (a) The nitrogen adsorption-desorption isotherm calculation shows that the specific surface area of ​​ZIF-8 is 1904 m 2 / g, the specific surface area of ​​UA@ZIF-8 is 611 m 2 / g, the specific surface area of ​​5-FU&UA@ZIF-8 is 218 m 2 / g, the specific surface area decreases successively. Figure 3 (b) The pore size distribution of ZIF-8 shows two types of pores in the range of 5-8 Å. After UA loading, the pore size and space are significantly reduced, and after 5-FU adsorption, the pores are further reduced. In summary, the pore size and space of UA@ZIF-8 match well with the size of 5-FU, and the pore space of ZIF-8 is reasonably utilized by the pore space partitioning method (PSP), and UA and 5-FU are successfully encapsulated.

[0059] Experimental Example 4

[0060] Determination of drug loading of products

[0061] Experimental steps:

[0062] Take 5 mg of 5-FU&UA@ZIF-8 from Example 1, digest it with 15 μL of hydrochloric acid to destroy the metal organic framework, make it dilute to 5 mL with methanol, and then dilute it 10 times. Use an ultraviolet spectrophotometer to scan the entire band, amplify the characteristic absorption area of ​​5-fluorouracil at a wavelength of 265 nm, and then calculate the drug loading amount based on the standard curve of 5-fluorouracil.

[0063] Take 5 mg of 5-FU&UA@ZIF-8 from Example 1, digest it with 15 μL of hydrochloric acid to destroy the metal organic framework, dilute it to 5 mL with methanol, take 0.5 mL and heat to dryness, and evaporate the solvent. Add 0.3 mL of 5% vanillin-glacial acetic acid solution and 0.7 mL of perchloric acid, and heat it in a 60°C water bath for 10 min. Take it out and cool it, then dilute it to 5 mL with acetic acid. Use a full-band scan of the ultraviolet spectrophotometer to amplify the characteristic absorption area of ​​ursolic acid at a wavelength of 545 nm, and then calculate the drug loading according to the standard curve of ursolic acid.

[0064] The drug loading calculation formula is as follows:

[0065]

[0066] Wherein, (wt%) is the drug loading, m is the mass of 5-fluorouracil or ursolic acid loaded into the metal organic framework, and M is the total mass of the products prepared in the examples and comparative examples.

[0067] Experimental results:

[0068] Depend on Figure 4 Calculation of the UV absorption spectrum after hydrochloric acid destruction shows that the UA loading in 5-FU&UA@ZIF-8 is 12.80 wt%, and the 5-FU loading is 11.20 wt%.

[0069] Experimental Example 5

[0070] Experimental steps:

[0071] The 5-FU&UA@ZIF-8 of Example 1 and the 5-FU@ZIF-8 of Comparative Example 2 were subjected to drug sustained release experiments in PBS at pH 7.4, 6.5 and 5.0, respectively. Specifically, 5 mg of the samples of the embodiment and the comparative example were taken respectively, and sustained release experiments were carried out in 10 mL PBS. A certain volume of supernatant was taken at different time intervals (0 min, 15 min, 30 min, 60 min, 100 min, 200 min, 300 min, 400 min, 500 min, 600 min, 700 min), and the possible trace precipitate was removed by centrifugation, and then the PBS corresponding to the pH of the taken volume was supplemented, and the possible precipitate was moved back to the sustained release solution. The absorbance was measured at a wavelength of 265 nm, and the drug release amount was calculated according to the standard curve of 5-fluorouracil.

[0072] The 5-FU&UA@ZIF-8 of Example 1 was subjected to drug sustained release experiments in 7.4, 6.5 and 5.0 PBS, respectively. Specifically, 5 mg of the samples of the example and the comparative example were taken and the sustained release experiments were carried out in 10 mL PBS. A certain volume of supernatant was taken at different time intervals (0h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h), and the possible trace precipitate was removed by centrifugation. After the solution was evaporated to dryness, 5% vanillin-acetic acid solution, perchloric acid and other color developing agents were added. The absorbance was measured at a wavelength of 545 nm, and the drug release amount was calculated according to the standard curve of ursolic acid.

[0073] Experimental results:

[0074] Depend on Figure 5 (b) It can be seen that 5-FU@ZIF-8 does not have the controlled release performance of 5-FU. Figure 5 (c) and Figure 5(d) It can be seen that 5-FU&UA@ZIF-8 has the ability to control the release of 5-FU and UA drugs, and more drugs are released under acidic conditions of pH 5.0 and pH 6.5, and less drugs are released under neutral conditions.

[0075] Experimental Example 6

[0076] Cytotoxicity assay

[0077] Experimental steps:

[0078] Preparation of experimental solution for ZIF-8 group: ZIF-8 was dissolved in 1640 medium, and 1640 medium was used to prepare ZIF-8 experimental solutions with concentrations of 5, 10, 20, 30, and 40 μg / mL;

[0079] Preparation of experimental solution for 5-FU@ZIF-8 group: 5-FU@ZIF-8 was dissolved in 1640 medium, and 5-FU@ZIF-8 experimental solutions with concentrations of 5, 10, 20, 30, and 40 μg / mL were prepared using 1640 medium;

[0080] Preparation of experimental solution for UA@ZIF-8 group: UA@ZIF-8 was dissolved in 1640 medium, and UA@ZIF-8 experimental solutions with concentrations of 5, 10, 20, 30, and 40 μg / mL were prepared using 1640 medium;

[0081] Preparation of experimental solution for 5-FU&UA@ZIF-8 group: 5-FU&UA@ZIF-8 was dissolved in 1640 culture medium, and 5-FU& UA@ZIF-8 experimental solutions with concentrations of 5, 10, 20, 30, and 40 μg / mL were prepared using 1640 culture medium.

[0082] Take 4T1 cells in the logarithmic growth phase and in good growth condition, perform cell digestion, and remove the original culture medium. First, add 2 mL of PBS solution to wash twice, add 1 mL of trypsin and place in the incubator for digestion for 4 min. After digestion, add 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% double antibody) to terminate digestion. Centrifuge at 1200 r / min for 5 min, remove the supernatant, add 9 mL of complete culture medium to it, blow evenly, and count the cells. Prepare a cell suspension according to experimental requirements and add 1×10 per well. 4Cells were plated in 96-well plates and cultured in an incubator for 24 h. After the cells adhered to the wall, the original culture medium was removed, and 200 μL of 1640 culture medium solution, ZIF-8 experimental solution, 5-FU@ZIF-8 experimental solution, UA@ZIF-8 experimental solution, and 5-FU&UA@ZIF-8 experimental solution were added to each well. After the addition of drugs, the plates were placed back in the incubator for 6 h. After the culture was completed, the drug-containing culture medium was aspirated, and 100 μL of 10% CCK-8 reagent was added to each well and the plates were placed in a 37 ℃ incubator for 2 h. Finally, the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader, and the data were recorded. The corresponding cell survival rate was calculated using the cell survival rate calculation formula, and a line graph was drawn with the cell survival rate as the y-axis and the drug concentration as the x-axis.

[0083] The cell survival rate formula is as follows:

[0084]

[0085] Experimental results:

[0086] Depend on Figure 6 It can be seen that at the maximum concentration of 40 μg / mL, only 20% of 4T1 cells died after ZIF-8 and cells were co-incubated for 6 hours, indicating that ZIF-8 has certain biocompatibility. After drug loading, 5-FU@ZIF-8, UA@ZIF-8 and 5-FU&UA@ZIF-8 showed obvious dose-dependent inhibition on cells, and compared with 5-FU@ZIF-8 and UA@ZIF-8 loaded with a single anticancer drug, 5-FU&UA@ZIF-8 had a stronger killing ability on 4T1 cells. This is because the synergistic anticancer effect of UA and 5-FU led to a significant decrease in cell viability, which also has great potential for overcoming drug resistance.

[0087] Experimental Example 7

[0088] Cellular uptake experiments

[0089] Experimental steps:

[0090] Preparation of experimental solution of 5-FU&UA@ZIF-8 modified with rhodamine B: 4 mg of rhodamine B was ultrasonically dissolved in 10 mL of methanol, 50 mg of 5-FU&UA@ZIF-8 was added, and magnetic stirring was performed for 12 h. Centrifuge at 10000 r / min for 10 min, the supernatant was discarded, methanol was added for washing, centrifugation and washing were repeated until the supernatant became colorless and transparent, and then dialyzed with a dialysis bag for 12 h. Finally, centrifuge, discard the supernatant, dry in an oven at 50°C overnight, and take out the pink crystalline powder in the centrifuge tube to obtain the sample of 5-FU&UA@ZIF-8 modified with rhodamine B (actually 24.5 mg was obtained). 5-FU&UA@ZIF-8 modified with rhodamine B was dissolved in 1640 culture medium, and a 20 μg / mL experimental solution of 5-FU& UA@ZIF-8 modified with rhodamine B was prepared using 1640 culture medium.

[0091] Take 4T1 cells in the logarithmic growth phase and in good growth condition, perform cell digestion, and remove the original culture medium. First, add 2 mL of PBS solution to wash twice, add 1 mL of trypsin and place in the incubator for digestion for 4 min. After digestion, add 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% double antibody) to terminate digestion. Centrifuge at 1200 r / min for 5 min, remove the supernatant, add 8 mL of complete culture medium to it, blow evenly, and count the cells. Prepare a cell suspension according to experimental requirements and place 5×10 per well. 4 Cells were plated in a 24-well plate and placed in an incubator for 24 h. After the cells adhered to the wall, the original culture medium was removed, and 1 mL of 5-FU&UA@ZIF-8 experimental solution modified with rhodamine B was added to each well. After the addition of the drug, the cells were placed back in the incubator for 0 h, 0.5 h, 2 h, and 4 h. After the culture was completed, the drug-containing culture medium was aspirated, washed 3 times with 1 mL PBS, and then 1 mL of 4% paraformaldehyde solution was added to each well to fix the cells at room temperature for 20 min. After fixation, the cells were washed twice with 1 mL PBS, and 100 μL of a certain concentration of nuclear dye DAPI staining solution was added to each well. The cells were stained in the dark at 37°C for 15 min, and washed twice with PBS. Finally, the cell phagocytosis of nanomaterials at different times was observed using a fluorescent inverted microscope.

[0092] Experimental results:

[0093] Depend on Figure 7 It can be seen that as time goes by, the red fluorescence in the 4T1 cells gradually brightens, verifying the effective uptake of 5-FU&UA@ZIF-8 materials by 4T1 cells, which to a great extent satisfies the effect of releasing drugs in cells after cells take up the materials.

[0094] Experimental Example 8

[0095] Live-dead cell imaging experiments

[0096] Experimental steps:

[0097] Preparation of experimental solution for ZIF-8 group: ZIF-8 was dissolved in 1640 medium, and 1640 medium was used to prepare a ZIF-8 experimental solution with a concentration of 20 μg / mL;

[0098] Preparation of experimental solution for 5-FU@ZIF-8 group: 5-FU@ZIF-8 was dissolved in 1640 medium, and 1640 medium was used to prepare 5-FU@ZIF-8 experimental solution with a concentration of 20 μg / mL;

[0099] Preparation of experimental solution for UA@ZIF-8 group: UA@ZIF-8 was dissolved in 1640 medium, and 1640 medium was used to prepare UA@ZIF-8 experimental solution with a concentration of 20 μg / mL;

[0100] Preparation of experimental solution for 5-FU&UA@ZIF-8 group: 5-FU&UA@ZIF-8 was dissolved in 1640 culture medium, and 1640 culture medium was used to prepare a 5-FU& UA@ZIF-8 experimental solution with a concentration of 20 μg / mL.

[0101] Take 4T1 cells in the logarithmic growth phase and in good growth condition, perform cell digestion, and remove the original culture medium. First, add 2 mL of PBS solution to wash twice, add 1 mL of trypsin and place in the incubator for digestion for 4 min. After digestion, add 4 mL of complete culture medium (containing 10% fetal bovine serum and 1% double antibody) to terminate digestion. Centrifuge at 1200 r / min for 5 min, remove the supernatant, add 8 mL of complete culture medium to it, blow evenly, and count the cells. Prepare a cell suspension according to experimental requirements and place 5×10 per well. 4 The cells were plated in a small dish and placed in an incubator for 24 h. After the cells attached to the wall, the original culture medium was removed, and 200 μL of 1640 culture medium solution, ZIF-8 experimental solution, 5-FU@ZIF-8 experimental solution, UA@ZIF-8 experimental solution, and 5-FU&UA@ZIF-8 experimental solution were added to each well. After the addition of drugs, the cells were placed in the incubator for 4 h. After the culture was completed, the drug-containing culture medium was aspirated, and 100 μL of a certain concentration of Calcein-AM (green for live cells) and PI (red for dead cells) mixed fluorescent dyes were added to each well. The cells were stained in the dark for 15 min at 37°C, washed twice with PBS, and the cells were placed under a fluorescence microscope to observe the fluorescence imaging.

[0102] Experimental results:

[0103] Depend on Figure 8 It can be seen that compared with the three groups of ZIF-8, 5-FU@ZIF-8, UA@ZIF-8, and 5-FU&UA@ZIF-8, the 5-FU&UA@ZIF-8 group still showed the most effective cell killing effect, as can be seen from the strong red fluorescence emitted by the stained dead cells, which is due to the synergistic anti-cancer effect of UA and 5-FU.

[0104] From the above experimental results, it can be seen that the zeolite imidazole metal organic framework material loaded with two anticancer drugs prepared by the pore space partitioning method (PSP) of the present invention has a high UA loading capacity (12.80 wt%) and 5-FU loading capacity (11.20 wt%). At the same time, by rationally selecting macromolecular UA to divide the pore space of ZIF-8, it has the ability to co-release 5-fluorouracil and ursolic acid drugs in response to pH, which can achieve the synergistic anticancer effect of 5-fluorouracil and ursolic acid, enhance the therapeutic effect on cancer tissue sites, and has certain clinical application potential.

Claims

1. A metal organic framework composite material for loading anticancer drugs using a pore space partitioning method, characterized in that: The loaded anticancer drugs are ursolic acid and 5-fluorouracil, and the molecular formula of the framework composite material is 5-FU&UA@ZIF-8; wherein the ursolic acid in the composite material is encapsulated in the pores of ZIF-8 and divides the pore space of ZIF-8, and the 5-fluorouracil is adsorbed into the remaining pores after being divided by ursolic acid.

2. The metal organic framework composite material according to claim 1, characterized in that: The drug loading of 5-fluorouracil is 11.2 wt%, and the drug loading of ursolic acid is 12.80 wt%. The ZIF-8 is a nano-sized metal organic framework material synthesized by a hydrothermal method using 2-methylimidazole as a ligand and zinc ions as metal ions.

3. A method for preparing the metal organic framework composite material according to claim 1 or 2, characterized in that: Use the following steps: (1) One-pot synthesis of UA@ZIF-8 zeolitic imidazole metal-organic framework composites loaded with ursolic acid; (2) Dispersing the UA@ZIF-8 synthesized in step (1) into an aqueous solution of 5-fluorouracil and stirring to obtain a zeolite imidazole metal organic framework composite material 5-FU&UA@ZIF-8 loaded with ursolic acid and 5-fluorouracil.

4. The preparation method according to claim 3, characterized in that: The "one-pot method" described in step (1) is prepared by a hydrothermal method; the specific steps are: dissolving zinc nitrate hexahydrate and ursolic acid in anhydrous methanol by ultrasonication, dissolving 2-methylimidazole in a sodium hydroxide aqueous solution, mixing the two thoroughly, stirring at room temperature, collecting the product after the reaction, washing, and obtaining UA@ZIF-8.

5. The preparation method according to claim 4, characterized in that: The stirring speed is 500-600 r / min, and the stirring time is 0.5-0.6 hour.

6. The preparation method according to claim 4, characterized in that: The mass ratio of zinc nitrate hexahydrate to ursolic acid is 5:1, and the concentration of zinc nitrate hexahydrate in anhydrous methanol is 15 mg / mL; the concentration of 2-methylimidazole in sodium hydroxide aqueous solution is 33 mg / mL, and the pH value of the sodium hydroxide aqueous solution is 12; wherein the volume ratio of anhydrous methanol to sodium hydroxide aqueous solution is 1:

1.

7. The preparation method according to claim 3, characterized in that: The UA@ZIF-8 is dispersed in an aqueous solution of 5-fluorouracil and stirred, and the UA@ZIF-8 absorbs 5-fluorouracil by physical adsorption.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the UA@ZIF-8 and 5-fluorouracil is 5:

8.

9. The preparation method according to claim 3, characterized in that: The concentration of 5-fluorouracil in the aqueous solution of 5-fluorouracil is 5-6 mg / mL.

10. The preparation method according to claim 3, characterized in that: The UA@ZIF-8 synthesized in step (1) was dispersed in an aqueous solution of 5-fluorouracil and stirred at a stirring speed of 300-500 r / min for 24 hours at room temperature.

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