Preparation method and application of zeolite imidazole frame-8 nano-drug delivery system

Abstract

The invention discloses a preparation method and an application of an iron-doped zeolite imidazole skeleton nano-drug delivery system (GOx / MTT coated Fe-ZIF-8) coated with glucose oxidase (GOx) and thiazole blue (MTT). The GOx / MTT coated Fe-ZIF-8 is decomposed to release Fe < 2 + >, GOx and MTT in an acidic environment of tumor cells. The released GOx further enhances the acid environment in the tumor while realizing hunger treatment, and damages the pentose phosphate pathway to cause insufficient supply of intracellular NADPH (Nicotinamide Adenine Dinucleotide Phosphate). Subsequently, the free MTT reacts with the NADPH to force the NADPH to be depleted and homocystine to be excessively uptake, so that disulfide in the cells is stressed to damage the activity and function of corresponding protein, and the disulfide death of the cells is caused. In addition, excessive H2O2 and Fe < 2 + > in cells are subjected to a Fenton reaction to generate a large amount of reactive oxygen species (ROS) to destroy intracellular oxidation-reduction balance, so that chemical kinetic treatment is realized. According to the nano-drug delivery system, targeted delivery of tumor cells is achieved, multi-mode collaborative treatment of hunger, disulfur death and chemical kinetics is achieved, toxic and side effects of treatment are reduced, and the tumor treatment effect is improved.

Description

Technical Field

[0001] The present invention belongs to the field of functional nanomaterials, and particularly relates to a preparation method of an iron-doped zeolitic imidazolate framework nanodrug delivery system loaded with glucose oxidase and thiazolyl blue responsive to pH and its application in multimodal synergistic cancer therapy. Background Art

[0002] Malignant tumors have become a major disease seriously threatening human life and health. Modern medicine has made remarkable progress in the early diagnosis and treatment of cancer, but there are still problems such as tumor drug resistance and strong side effects. The development of emerging fields such as molecular biology, genomics, and immunotherapy has provided new directions for the efficient and precise treatment of tumors. Multimodal synergistic strategies such as starvation therapy and chemodynamic therapy are gradually becoming important means to deal with malignant tumors due to their advantages in high targeting, low side effects, and reducing tumor drug resistance. Based on the acidic cellular environment of tumor cells, a pH-responsive nanomaterial is designed to achieve precise drug delivery and controlled release at the tumor site.

[0003] In recent years, zeolitic imidazolate framework (ZIF) materials with good biocompatibility have attracted extensive attention of researchers. First, ZIF has a high specific surface area and large pore volume, which can load a variety of anti-tumor drugs and increase drug concentration; second, ZIF has acid-responsive decomposition characteristics, enhancing drug targeting and biocompatibility; in addition, ZIF can achieve slow drug release, prolong the action time of drugs in vivo, and reduce the dosing frequency; finally, ZIF can be combined with other nanomaterials such as metal nanoparticles and quantum dots to achieve the integration of diagnosis and treatment. Therefore, ZIF materials show great potential in the field of tumor precision therapy.

[0004] Disulfidptosis is a completely new form of programmed cell death proposed in 2023, independent of existing forms such as apoptosis, ferroptosis, necroptosis, and cuproptosis. It is a rapid death caused by disulfide stress caused by excessive accumulation of cystine in cells. Its main mechanism involves the high expression of solute carrier SLC7A11 and glucose starvation. Under glucose-deprived conditions, cells with high expression of SLC7A11 will take in a large amount of cystine, but due to insufficient NADPH supply, cystine cannot be reduced to cysteine in time, resulting in abnormal accumulation of disulfides in cells. These accumulated disulfides will interfere with the organization between actin cytoskeletal proteins, ultimately leading to the collapse of the actin cytoskeleton and cell death, thereby inhibiting tumor growth. Using zeolitic imidazolate framework as a nanocarrier, by loading thiazolyl blue and glucose oxidase, multimodal synergistic therapy of disulfidptosis, starvation therapy, and chemodynamics responsive to the tumor microenvironment is achieved, improving the therapeutic effect of tumors while reducing the toxicity to normal cells, and having broad application prospects in the field of tumor treatment. Summary of the Invention

[0005] To solve the problems of weak targeting and strong toxic side effects of traditional tumor treatment methods, the present invention constructs a tumor microenvironment-responsive iron-doped zeolitic imidazolate framework (Fe-ZIF-8) based on iron-doped zeolitic imidazolate framework, and a nano-drug delivery system (GOx / MTT@Fe-ZIF-8) is prepared by loading thiazolyl blue (MTT) and glucose oxidase (GOx) on Fe-ZIF-8. This nano-drug delivery system efficiently aggregates in tumor cells and releases Fe 2+ , GOx and thiazolyl blue through pH-responsive decomposition. The released GOx decomposes intracellular glucose to generate gluconic acid and H2O2, while performing starvation therapy, further enhancing the acidic environment in tumors, and disrupting the pentose phosphate pathway to cause insufficient intracellular NADPH supply. Subsequently, the free MTT reacts with NADPH, forcing the depletion of NADPH and the excessive uptake of homocysteine, resulting in the disruption of the activity and function of corresponding proteins due to intracellular disulfide stress, causing cell disulfide death. In addition, the excessive H2O2 in tumor cells reacts with the released Fe 2+ to undergo a Fenton reaction, generating a large amount of reactive oxygen species (ROS) to disrupt the intracellular redox balance and achieve the purpose of chemodynamic therapy. This drug delivery system responds to the tumor microenvironment, realizing multi-modal synergistic therapy of tumor starvation / disulfide death / chemodynamic, reducing toxic side effects while improving the therapeutic effect on tumors.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A preparation method and application of a tumor microenvironment-responsive zeolitic imidazolate framework-8 nano-drug delivery system are as follows:

[0007] (1) Preparation of the nano-drug delivery system: Dissolve 2-methylimidazole (2-MIM) in deionized water, add GOx and MTT thereto, and after complete dissolution, add a mixed aqueous solution of FeSO4·7H2O and Zn(NO3)2·6H2O, stir at room temperature overnight, and obtain iron-doped zeolitic imidazolate framework-8 nanoparticles loaded with GOx and MTT after centrifugation, washing and freeze-drying, denoted as GOx / MTT@Fe-ZIF-8;

[0008] The preferred manner of the above preparation method is as follows:

[0009] In the step (1), the molar ratio of FeSO4·7H2O, Zn(NO3)2·6H2O and 2-methylimidazole is 1:19:1500;

[0010] In the step (1), the stirring time is 12 hours and the freeze-drying time is 18 h.

[0011] (2) Characterization of GOx / MTT@Fe-ZIF-8: The prepared GOx / MTT@Fe-ZIF-8 nano-delivery system was characterized by scanning electron microscopy (SEM) and particle size analysis. The amounts of GOx and MTT loaded in GOx / MTT@Fe-ZIF-8 were calculated by ultraviolet-visible absorption spectroscopy; the slow-release of GOx / MTT@Fe-ZIF-8 in buffer solution (pH = 5.5) was studied by ultraviolet-visible absorption spectroscopy; the catalytic effect of glucose oxidase on glucose before and after loading was verified by detecting the pH change of glucose solution; the therapeutic effect of GOx / MTT@Fe-ZIF-8 on tumor cells (MCF-7) was verified by AnnexinV-FITC / PI.

[0012] Advantages of the present invention:

[0013] (1) Under the stimulation of the tumor acidic microenvironment, the nano-drug delivery system prepared in the present invention releases glucose oxidase to achieve starvation therapy for tumor cells, and at the same time leads to an increase in the intracellular H2O2 concentration and the loss of the source of NADPH.

[0014] (2) Under the stimulation of the tumor acidic microenvironment, the released Fe 2+ in the nano-drug delivery system prepared in the present invention reacts with H2O2 to generate ·OH to achieve chemodynamic therapy for tumors.

[0015] (3) Under the stimulation of the tumor acidic microenvironment, the nano-drug delivery system prepared in the present invention releases MTT, consumes NADPH leading to NADPH depletion, and forces cellular disulfide stress to achieve disulfidptosis of tumor cells.

[0016] (4) The nano-drug delivery system prepared in the present invention achieves multimodal synergistic therapy of disulfidptosis, starvation therapy and chemodynamic therapy for tumor cells through cascade reactions, increasing the therapeutic effect. Description of the drawings

[0017] Figure 1 It is a schematic diagram of the preparation of the GOx / MTT@Fe-ZIF-8 nano-carrier and multimodal therapy for tumors described in the present invention.

[0018] Figure 2 It is the SEM and particle size distribution diagram of the GOx / MTT@Fe-ZIF-8 nano-material.

[0019] Figure 3 It is a graph showing the relationship between the release amount of GOx enzyme and time of GOx / MTT@Fe-ZIF-8 in buffer solution at pH = 5.5.

[0020] Figure 4Relationship diagram of the release amount of Mmt enzyme of GOx / MTT@Fe-ZIF-8 and time in pH = 5.5 buffer solution.

[0021] Figure 5 Fe released by GOx / MTT@Fe-ZIF-8 in pH = 5.5 buffer solution 2+ Relationship diagram with time.

[0022] Figure 6 It is the survival rate of tumor cells (MCF-7) detected by MTT method after 24h of cell incubation under different nanoparticle incubations. Specific implementation manner

[0023] The present invention will be further described in conjunction with the embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0024] Example 1

[0025] Preparation of GOx / MTT@Fe-ZIF-8: Dissolve 1.784 g of 2-MIM in 4 mL of deionized water, then add 10 mg of Mmt and 6 mg of GOx. After stirring for 5 min, 56.8 mg of Zn(NO3)2·6H2O dissolved in 0.4 mL of water and 3.0 mg of FeSO4·7H2O were rapidly injected into the above solution under vigorous stirring at room temperature. After continuously reacting for 12 h under stirring conditions, separation was carried out by centrifugation (13200 rpm, 10 min), washed twice with 10% ethanol (v / v), and washed twice with deionized water. The obtained solid was placed in an environment at -10 °C for 24 h, and then freeze-dried to obtain a light brown powder.

[0026] Example 2

[0027] Use ultraviolet-visible absorption spectroscopy to evaluate the slow release of GOx and MTT in GOx / MTT@Fe-ZIF-8 in buffer solution (pH = 5.5) over time. The slow release of GOx and MTT over time can be evaluated by measuring the ultraviolet absorption intensity of the supernatant and according to the standard calibration curve. Take 5 mg of the GOx / MTT@Fe-ZIF-8 nanoparticles prepared in Example 1 and disperse them in 5 mL of PBS buffer solution (pH = 5.5). Take 200 μL of the supernatant at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and measure its absorption intensity at 280 nm. Draw a curve of the release amount of GOx versus time according to the standard calibration curve (attached Figure 3 ). The release curve of MTT (380 nm) can be obtained by the same method (attached Figure 4 ). As shown in attached Figure 3 and attached Figure 4As shown, GOx and MTT showed similar release patterns, with 90% release achieved at 10 h, indicating that the prepared nanomaterial GOx / MTT@Fe-ZIF-8 has the property of weak acid-responsive release and can effectively release GOx and MTT in the tumor microenvironment.

[0028] Example 3

[0029] The release ability of GOx / MTT@Fe-ZIF-8 in buffer solution (pH = 5.5) was evaluated by ultraviolet-visible absorption spectroscopy for Fe 2+ Using 1,10-phenanthroline as the chromogenic agent for Fe 2+ detection, the content of Fe in the solution was quantitatively analyzed by detecting the absorption intensity at 512 nm of the mixed solution. The same method as in Example 2 was used to prepare a 1 mg / mL GOx / MTT@Fe-ZIF-8 solution (pH = 5.5), and 1,10-phenanthroline was added. Subsequently, 200 μL of the supernatant was taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h respectively, and its absorption intensity at 512 nm was measured. According to the standard calibration curve, the change curve of Fe 2+ release amount with time was plotted. As shown in the appendix 2+ The release rate gradually increased, with a release rate of about 50% at 1 h and almost complete release after 12 h. Figure 5 As shown, the release rate gradually increased, with a release rate of about 50% at 1 h and almost complete release after 12 h.

[0030] Example 4

[0031] The study on the activity of GOx before and after coating was carried out by monitoring the change of pH of glucose solution. Free GOx and GOx / MTT@Fe-ZIF-8 with the same GOx content were respectively dissolved in equal volumes of glucose solution (2 mg / mL), and the pH value of the solution was monitored by a pH meter at regular intervals. Free GOx had good enzyme activity within 3 h and could effectively decompose glucose to produce gluconic acid, but as a large amount of gluconic acid was generated, the pH of the solution gradually decreased and GOx gradually lost its activity. Under the same conditions, compared with free GOx, the activity of GOx / MTT@Fe-ZIF-8 was significantly reduced within the same time because the pore structure of Fe-ZIF-8 might cause certain hindrance to the diffusion of substrates (glucose) and products (H2O2), resulting in a decrease in apparent activity. However, due to the protection of the Fe-ZIF-8 shell, its action time was greatly extended, and its stability, reusability, and co-catalytic ability were significantly better than those in the free state.

[0032] Example 5

[0033] The therapeutic effect of the nanomaterial GOx / MTT@Fe-ZIF-8 prepared in Example 1 on tumors was verified using Annexin V-FITC / PI. MCF-7 cells were seeded in 6-well plates and incubated in a cell culture incubator containing 5% CO2 at 37 °C for 12 h. The original cell culture medium was aspirated, and cell culture media containing different amounts (0 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL) of GOx / MTT@Fe-ZIF-8 were added to each treatment well. After incubation for 24 h, the cells were digested, washed with PBS, centrifuged, and then resuspended in PBS solution. 5 μL of Annexin V-FITC and 10 μL of PI solution were added, and incubation was continued for 1 h. After incubation, 400 μL of PBS solution was added, and after mixing, the apoptosis of cells in each treatment group was analyzed by flow cytometry. The results are as Figure 6 shown.

Claims

1. A tumor microenvironment-responsive zeolitic imidazolate framework nanodrug delivery system that can be used for tumor disulfidptosis, starvation death, and chemodynamic therapy. The zeolitic imidazolate framework nanodrug delivery system uses iron-doped zeolitic imidazolate framework-8 as a carrier and encapsulates glucose oxidase (GOx) and thiazolyl blue (MTT).

2. A preparation method of a zeolitic imidazolate framework-8 nanomedicine delivery system, characterized in that, The preparation steps are as follows: (1) Dissolve 2-methylimidazole in deionized water, add GOx and MTT thereto, and after complete dissolution, add an aqueous mixed solution of FeSO4·7H2O and Zn(NO3)2·6H2O. Stir at room temperature, and after centrifugation, washing, and freeze-drying, obtain iron-doped zeolitic imidazolate framework-8 nanoparticles encapsulating GOx and MTT, denoted as GOx / MTT@Fe-ZIF-8.

3. Application of the nanodrug delivery system described in claim 1 in tumor treatment.

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