A particle beam irradiation-induced disordered MOF nanozyme, its preparation method and application

Abstract

This invention belongs to the field of nanozymes and biomedicine, and provides a particle beam irradiation-induced disordered MOF nanozyme, its preparation method, and its application. Ferric nitrate nonahydrate, trimesic acid, and water are mixed and reacted to obtain pure-phase crystalline MIL-100(Fe) powder. The pure-phase crystalline MIL-100(Fe) powder is then mixed with water and irradiated to obtain the particle beam irradiation-induced disordered MOF nanozyme. This invention achieves precise control of the MOF crystal structure through high-energy electron beam irradiation. By locally breaking organic ligands and inducing Fe-O coordination bond distortion, a stable crystalline-amorphous mixed-state structure is formed. While retaining the advantages of the three-dimensional pore structure of MIL-100(Fe), an appropriate amount of amorphous regions and unsaturated Fe coordination sites are introduced, significantly increasing the amount of unsaturated Fe. 3+ The increased number of active sites resulted in a significant improvement in the activity of multiple enzymes.

Description

Technical Field

[0001] This invention relates to the field of nanozymes and biomedicine, and in particular to a particle beam irradiation-induced disordered MOF nanozyme, its preparation method, and its application. Background Technology

[0002] Nanozymes are a class of nanomaterials possessing the catalytic activity of natural enzymes. Compared to natural enzymes, they offer advantages such as high stability, low cost, ease of storage, and functionalization, demonstrating enormous application potential in fields such as biomedicine, environmental remediation, and catalysis. Among them, iron-based metal-organic framework (Fe-MOF) nanozymes have become a research hotspot in the field of anti-tumor nanocatalysis medicine due to their tunable pore structure, abundant Fe active sites, and good biocompatibility.

[0003] However, pure crystalline Fe-MOF nanozymes have significant drawbacks: their metal nodes are fully coordinated with organic ligands, and a large number of catalytic active sites are encapsulated within the framework, making it difficult for them to contact the substrate and resulting in low enzyme activity. To address this issue, existing technologies often employ high-temperature calcination to prepare MOF derivatives. However, high-temperature treatment leads to the complete collapse of the MOF framework and severe particle aggregation, which not only destroys its unique pore structure but also causes the encapsulation and inactivation of active sites.

[0004] In recent years, amorphization modification has been proven to be an efficient strategy for enhancing the catalytic activity of metal-organic framework (MOF) nanozymes. Amorphous MOFs possess a structural characteristic of long-range disorder and short-range order, exposing a large number of metal coordination unsaturated sites, significantly enhancing enzyme-like catalytic performance. However, traditional amorphization methods such as mechanical ball milling and high-temperature heat treatment have significant limitations: ball milling easily causes framework breakage and particle agglomeration, while high-temperature treatment easily leads to the decomposition of organic ligands and collapse of the pore structure; both make it difficult to precisely control the degree of amorphization. Insufficient amorphization results in limited exposure of active sites, while excessive amorphization destroys the intrinsic structure of MOFs and reduces material stability.

[0005] Therefore, overcoming the problems caused by amorphization modification has become a research direction for nanozymes. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies in the prior art and provide a particle beam irradiation-induced disordered MOF nanozyme, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing particle beam irradiation-induced disordered MOF nanozymes, comprising the following steps: (1) After mixing ferric nitrate nonahydrate, trimesic acid and water, a reaction was carried out to obtain pure phase crystalline MIL-100(Fe) powder; (2) The pure phase crystalline MIL-100(Fe) powder was mixed with water and then irradiated to obtain the particle beam irradiation induced disordered MOF nanozyme.

[0008] As a preferred option, the ratio of ferric nitrate nonahydrate, trimesic acid and water in step (1) is 1.4~1.6 mol: 1 mol: 2.5~3.5 L.

[0009] Preferably, the reaction temperature in step (1) is 100~120℃ and the time is 10~15h.

[0010] Preferably, in step (2), the ratio of pure phase crystalline MIL-100(Fe) powder to water is 50~70mg:20~40mL.

[0011] Preferably, the irradiation dose in step (2) is 25~200kGy and the time is 5~10min.

[0012] This invention provides a method for preparing particle beam irradiation-induced disordered MOF nanozymes, resulting in particle beam irradiation-induced disordered MOF nanozymes.

[0013] The present invention also provides the application of the particle beam irradiation-induced disordered MOF nanozyme in the preparation of antitumor drugs.

[0014] This invention provides a method for preparing particle beam irradiation-induced disordered MOF nanozymes, comprising the following steps: mixing ferric nitrate nonahydrate, trimesic acid, and water and reacting the mixture to obtain pure-phase crystalline MIL-100(Fe) powder; mixing the pure-phase crystalline MIL-100(Fe) powder with water and then irradiating the mixture to obtain the particle beam irradiation-induced disordered MOF nanozymes. This invention achieves precise control of the MOF crystal structure through high-energy electron beam irradiation. By locally breaking organic ligands and inducing Fe-O coordination bond distortion, a stable crystalline-amorphous mixed-state structure is formed. While retaining the advantages of the three-dimensional pore structure of MIL-100(Fe), an appropriate amount of amorphous regions and unsaturated Fe coordination sites are introduced, significantly increasing the amount of unsaturated Fe. 3+ The increased number of active sites resulted in a significant improvement in the activity of multiple enzymes.

[0015] This invention pioneers a composite amorphization process using high-energy electron beam irradiation, employing mild preparation conditions that avoid particle agglomeration and framework collapse caused by high-temperature calcination. By varying the irradiation dose, the degree of amorphization can be precisely controlled, achieving the optimal ratio of crystalline and amorphous phases. The resulting particle beam irradiation-induced disordered MOF nanozymes possess both the structural stability of crystalline materials and the high catalytic activity of amorphous materials. The crystalline phase retains three-dimensional mesoporous channels, facilitating substrate diffusion and transport; the amorphous phase exposes numerous unsaturated Fe coordination sites, while Fe... 2+ / Fe 3+ The presence of mixed valence states promotes redox cycles, and its peroxidase-like (POD), glutathione peroxidase-like (GPx), catalase-like (CAT), and superoxide dismutase-like (SOD) activities are significantly enhanced compared to pure crystalline MIL-100 (Fe).

[0016] The particle beam irradiation-induced disordered MOF nanozyme provided by this invention simultaneously exists in crystalline and amorphous phases. The crystalline phase retains the three-dimensional mesoporous channel structure of MIL-100 (Fe), while the amorphous phase is formed by the breakage of some organic ligands and the distortion of Fe-O bonds. The Fe element in the material is primarily Fe2+. 2+ / Fe 3+ Valence states exist, and the number of coordination sites for unsaturated Fe increases compared to pure crystalline MIL-100(Fe).

[0017] The particle beam irradiation-induced disordered MOF nanozyme provided by this invention is applied in the preparation of anti-tumor drugs, exerting its effects through chemokinetic therapy combined with immunotherapy: on the one hand, its excellent peroxidase-like (POD) activity can catalyze the generation of highly oxidizing hydroxyl radicals from high concentrations of H2O2 in the tumor microenvironment (…). The activity of glutathione peroxidase (GPx) can consume excess glutathione (GSH) in tumor cells, disrupting the cellular redox balance and inducing ferroptosis. On the other hand, ferroptosis can further induce immunogenic cell death (ICD), promote the eversion of calreticulin (CRT) and the release of high-migration group box 1 (HMGB1), and upregulate PD-L1 expression in tumor cells, thereby significantly enhancing the therapeutic effect of immune checkpoint inhibitors. Furthermore, the nanozyme prepared in this invention has good biocompatibility, and in vitro and in vivo experiments have demonstrated that it can significantly inhibit the growth of HeLa human cervical cancer cells. Moreover, it achieves synergy between chemokinetics and immunotherapy by inducing ferroptosis and immunogenic cell death, providing a new approach for the development of highly efficient anti-tumor nanomedicines. Attached Figure Description

[0018] Figure 1 XRD patterns of different samples from Example 1 and Comparative Example 1; Figure 2TEM and HRTEM images of the samples from Comparative Example 1 and Comparative Example 2; Figure 3 TEM and HRTEM images of the Q-Fe-MOF (25 kGy) samples from Comparative Example 1 and Example 1; Figure 4 EXAFS characterization images of different samples from Example 1 and Comparative Example 1; Figure 5 Comparison chart of POD-like, GPx, CAT, and SOD activities of Comparative Example 1, Comparative Example 2, and Example 1 Q-Fe-MOF (25 kGy) samples; Figure 6 This is a comparison of the survival rates of HeLa cells after treatment with Q-Fe-MOF (25 kGy) in Comparative Example 1 and Example 1. Detailed Implementation

[0019] This invention provides a method for preparing particle beam irradiation-induced disordered MOF nanozymes, comprising the following steps: (1) After mixing ferric nitrate nonahydrate, trimesic acid and water, a reaction was carried out to obtain pure phase crystalline MIL-100(Fe) powder; (2) The pure phase crystalline MIL-100(Fe) powder was mixed with water and then irradiated to obtain the particle beam irradiation induced disordered MOF nanozyme.

[0020] In this invention, the preferred ratio of ferric nitrate nonahydrate, trimesic acid and water in step (1) is 1.4~1.6 mol: 1 mol: 2.5~3.5 L, more preferably 1.45~1.55 mol: 1 mol: 2.6~3.4 L, and even more preferably 1.48~1.52 mol: 1 mol: 2.8~3.2 L.

[0021] In this invention, after ferric nitrate nonahydrate and trimesic acid are completely dissolved, they are allowed to stand for a period of ≥2 hours, more preferably ≥3 hours, and even more preferably ≥4 hours; after the standing period, the reaction is carried out.

[0022] In this invention, the temperature of the reaction in step (1) is preferably 100~120℃, more preferably 105~115℃, and even more preferably 108~112℃; the time is preferably 10~15h, more preferably 11~14h, and even more preferably 12~13h.

[0023] In this invention, after the reaction in step (1) is completed, the product is naturally cooled to room temperature and collected by centrifugation. The centrifugation speed is preferably ≥8000 rpm, more preferably ≥9000 rpm, and more preferably ≥10000 rpm. The collected product is washed with anhydrous ethanol and deionized water, preferably 3 to 5 times. After washing, it is vacuum dried. The vacuum drying temperature is preferably 50 to 70°C, more preferably 55 to 65°C, and more preferably 58 to 62°C. The vacuum drying time is preferably ≥2 h, more preferably ≥3 h, and more preferably ≥4 h. After drying, pure phase crystalline MIL-100(Fe) powder is obtained.

[0024] In this invention, the preferred ratio of pure phase crystalline MIL-100(Fe) powder to water in step (2) is 50~70mg:20~40mL, more preferably 55~65mg:25~35mL, and even more preferably 58~62mg:28~32mL; after mixing evenly, the mixture is transferred to a PE plastic bag for the next step of irradiation treatment.

[0025] In this invention, the irradiation dose in step (2) is preferably 25~200kGy, more preferably 25~150kGy, and even more preferably 25~100kGy; the time is preferably 5~10min, more preferably 6~9min, and even more preferably 7~8min.

[0026] In this invention, after irradiation, the powder is collected and washed with anhydrous ethanol and deionized water, preferably 3 to 5 times; after washing, it is vacuum dried; the vacuum drying temperature is preferably 50 to 70°C, more preferably 55 to 65°C, and even more preferably 58 to 62°C; the vacuum drying time is preferably ≥2h, more preferably ≥3h, and even more preferably ≥4h; after drying, particle beam irradiation-induced disordered MOF nanozyme is obtained.

[0027] The present invention also provides a method for preparing particle beam irradiation-induced disordered MOF nanozymes, resulting in particle beam irradiation-induced disordered MOF nanozymes.

[0028] The present invention also provides the application of the particle beam irradiation-induced disordered MOF nanozyme in the preparation of antitumor drugs.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] (1) Synthesis of pure-phase crystalline MIL-100 (Fe): 6.060 g of ferric nitrate nonahydrate and 2.101 g of trimesic acid (Fe) were weighed. 3+(2) Dissolve BTC=1.5:1 in 30 mL of deionized water; (3) Stir until completely dissolved and transfer to a polytetrafluoroethylene-lined hydrothermal reactor, stand for 2 h, and then react at 110℃ for 12 h; (4) After the reaction is completed, cool naturally to room temperature, centrifuge at 8000 rpm to collect the product, wash with anhydrous ethanol and deionized water 5 times, and dry the obtained powder under vacuum at 60℃ for 2 h to obtain pure phase crystalline MIL-100(Fe) powder; (5) Take 60 mg of the above crystalline powder and disperse it in 30 mL of deionized water, mix evenly and transfer to a PE plastic bag; (6) Irradiate the powder with a 25-200 kGy high-energy electron beam for 5 min to amorphize it; (7) Wash the irradiated powder with anhydrous ethanol and deionized water alternately 4 times, and dry it under vacuum at 60℃ for 2 h to obtain particle beam irradiation induced disordered MOF nanozymes, denoted as Q-Fe-MOF(25 kGy) and Q-Fe-MOF(50 kGy). kGy), Q-Fe-MOF (100 kGy), Q-Fe-MOF (200kGy).

[0032] Comparative Example 1

[0033] Pure phase crystalline MIL-100(Fe): prepared by the method of steps (1)-(3) in Example 1, without electron beam irradiation, and denoted as C-Fe-MOF.

[0034] Comparative Example 2

[0035] Ball milling of fully amorphous MIL-100(Fe): Pure phase crystalline MIL-100(Fe) was placed in a ball mill jar and thoroughly ground with an agate mortar. Zirconia balls with a mass ratio of 1 / 30 were weighed and added to the ball mill jar. The ball mill jar was placed in a high-energy ball mill and ball milled at 350 rpm for 5 min, then stopped for 5 min, and the cycle was repeated three times. The ball-milled sample was then placed in an agate mortar and ground for 15 min, which was recorded as Q-Fe-MOF (15 min).

[0036] The XRD patterns of different samples from Example 1 and Comparative Example 1 are as follows: Figure 1 As shown, from Figure 1 It can be seen that as the irradiation dose increases from 25 kGy to 200 kGy, the characteristic diffraction peaks of MIL-100(Fe) decrease in intensity and broaden in shape at low doses (25 kGy and 50 kGy intensities), and Q-Fe-MOF(25 kGy) exhibits typical quasicrystalline characteristics.

[0037] TEM and HRTEM images of the samples from Comparative Example 1 and Comparative Example 2 are shown below. Figure 2 As shown, Figure 2In the image, a is the TEM image of the sample in Comparative Example 1, b is the HRTEM image of the sample in Comparative Example 1, c is the TEM image of the sample in Comparative Example 2, and d is the HRTEM image of the sample in Comparative Example 2. TEM and HRTEM images of the Q-Fe-MOF (25 kGy) samples from Comparative Example 1 and Example 1 are shown below. Figure 3 As shown, Figure 3 In the image, a is the TEM image of the sample of Comparative Example 1, b is the HRTEM image of the sample of Comparative Example 1, c is the TEM image of the Q-Fe-MOF (25 kGy) sample of Example 1, and d is the HRTEM image of the Q-Fe-MOF (25 kGy) sample of Example 1. from Figure 2 and Figure 3 As can be seen, the original C-Fe-MOF exhibits a regular octahedral single crystal morphology, and selected area electron diffraction (SAED) reveals a clear and regular lattice diffraction pattern. After ball milling and 25 kGy electron beam irradiation, the overall particle outline of the sample is still intact, with only a significant disordered thin layer forming at the particle edges. At the same time, the SAED pattern changes from a regular lattice to a diffuse scattering ring, which directly confirms that the material has achieved a structural transformation from a crystalline state to a quasi-crystalline state. This is quite different from the defects caused by mechanical ball milling, which easily leads to particle breakage, morphological damage, and severe agglomeration. Electron beam irradiation modification is more gentle and controllable, and can precisely induce local structural disorder and construct abundant defect active sites without destroying the overall particle morphology.

[0038] EXAFS characterization images of different samples from Example 1 and Comparative Example 1 are shown below. Figure 4 As shown, from Figure 4 The unique advantages of electron beam irradiation in regulating the Fe coordination environment are evident. According to the fitted parameters, the Fe-O coordination number in C-Fe-MOF is 6.15, indicating a highly saturated coordination state. After irradiation with a 25 kGy electron beam, the Fe-O coordination number in Q-Fe-MOF (25 kGy) decreased to 5.01, with the breakage of some Fe-O bonds creating highly active unsaturated Fe sites. Compared to mechanical ball milling, which results in no severe bond breakage or ligand loss, 25 kGy electron beam irradiation achieves precise regulation of the Fe-MOF coordination environment: while maintaining the Fe central trivalent state and overall framework stability, it introduces high-density unsaturated coordination defect sites by breaking some Fe-O coordination bonds without causing metal phase formation or framework collapse. This provides abundant active sites for subsequent enzyme-like catalytic reactions, while preserving the material's structural stability and pore integrity, demonstrating significant modification advantages.

[0039] Enzyme activity test

[0040] The following methods were used to determine POD-like activities: the 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric method; the GPx-like activities: the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) method; the CAT-like activities: the dissolved oxygen meter method; and the SOD-like activities: the nitroblue tetrazolium (NBT) reduction method. Test conditions: sample concentration 30 μg / mL, pH 4.0 (POD) or pH 7.4 (GPx, CAT, SOD). Test results are as follows: Figure 5 As shown, Figure 5 In the diagram, a is a comparison of POD-like activities, b is a comparison of GPx-like activities, c is a comparison of CAT-like activities, and d is a comparison of SOD-like activities; from Figure 5 It can be seen that the POD, GPx, CAT, and SOD activities of Q-Fe-MOF (25 kGy) are significantly higher than those of C-Fe-MOF and Q-Fe-MOF (15 min). Among them, the POD-like activity of Q-Fe-MOF (25 kGy) is the most significantly improved, which is attributed to the large amount of Fe after electron beam amorphization. 3+ Exposure of coordination unsaturated sites. Q-Fe-MOF (15 min) showed a significant decrease in enzyme activity due to framework collapse, pore disappearance, and aggregation of active sites.

[0041] HeLa cell experiments

[0042] Human cervical cancer cells (HeLa) were used as a tumor killing model, and human brain microvascular endothelial cells (HCMEC / D3) were used as normal cell controls. The in vitro cytotoxicity of C-Fe-MOF and Q-Fe-MOF (25 kGy) nanoplatforms was systematically evaluated using the CCK-8 assay. First, two types of cells in the logarithmic growth phase were seeded at an initial density of 8000 cells per well in 96-well plates and cultured for 24 h in high-glucose DMEM medium containing streptomycin (100 μg / mL), penicillin (100 μg / mL), and fetal bovine serum (10% FBS). After the cells were fully adhered and growing well, the original medium was aspirated, and fresh culture medium containing different concentration gradients (0, 50, 100, 200 μg / mL) of the target material was added and co-incubated for another 24 h. Then, 10 μL of CCK-8 detection solution was added to each well under light-protected conditions, and the wells were wrapped with aluminum foil and incubated for another 2 h. The absorbance (OD value) of each well was then measured using a multi-mode microplate reader at a wavelength of 450 nm. Finally, the absorbance was calculated using the formula [OD value]. 实验组 -OD 空白组 ] / [OD 对照组 -OD 空白组 ]×100%, calculate the relative cell survival rate.

[0043] The comparison of HeLa cell viability after treatment with Q-Fe-MOF (25 kGy) in Comparative Example 1 and Example 1 is shown in the figure below. Figure 6 As shown, from Figure 6 It can be seen that the original crystalline C-Fe-MOF exhibited a concentration-dependent inhibitory effect on the proliferation of both cell types, but the killing difference between tumor cells and normal cells was not significant: when the concentration reached 200 μg / mL, the survival rate of HeLa cells decreased to 31.2%, while the survival rate of HCMEC / D3 cells remained at 66.7%, indicating that the antitumor activity of C-Fe-MOF was limited and lacked obvious selectivity. In stark contrast, Q-Fe-MOF (25 kGy) showed significant tumor-selective killing ability: at the same concentration gradient, its inhibitory effect on the proliferation of HeLa cells was significantly enhanced, with a cell survival rate of only 7.8% at 200 μg / mL; while its toxicity to normal HCMEC / D3 cells was extremely low, with a cell survival rate as high as 59.6% at the same concentration.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing particle beam irradiation-induced disordered MOF nanozymes, characterized in that, Includes the following steps: (1) After mixing ferric nitrate nonahydrate, trimesic acid and water, a reaction was carried out to obtain pure phase crystalline MIL-100(Fe) powder; (2) The pure phase crystalline MIL-100(Fe) powder was mixed with water and then irradiated to obtain the particle beam irradiation induced disordered MOF nanozyme.

2. The method for preparing particle beam irradiation-induced disordered MOF nanozymes as described in claim 1, characterized in that, In step (1), the ratio of ferric nitrate nonahydrate, trimesic acid and water is 1.4~1.6 mol: 1 mol: 2.5~3.5 L.

3. The method for preparing particle beam irradiation-induced disordered MOF nanozymes as described in claim 2, characterized in that, The reaction in step (1) is carried out at a temperature of 100~120℃ for 10~15h.

4. The method for preparing particle beam irradiation-induced disordered MOF nanozymes as described in claim 3, characterized in that, In step (2), the ratio of pure phase crystalline MIL-100(Fe) powder to water is 50~70mg:20~40mL.

5. The method for preparing particle beam irradiation-induced disordered MOF nanozymes as described in claim 4, characterized in that, In step (2), the irradiation dose is 25~200kGy and the time is 5~10min.

6. The particle beam irradiation-induced disordered MOF nanozyme prepared by the method of any one of claims 1 to 5.

7. The application of the particle beam irradiation-induced disordered MOF nanozyme of claim 6 in the preparation of antitumor drugs.

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