Method for improving oxygen activation efficiency of nano zero-valent iron by using tetrahydrofuran as reaction medium and application thereof

Abstract

This invention discloses a method for synthesizing nano-zero-valent iron (nZVI) using tetrahydrofuran as a reaction medium to improve oxygen activation efficiency and its application. This invention introduces tetrahydrofuran into the preparation process of nano-zero-valent iron (nZVI), utilizing tetrahydrofuran to regulate the morphology of nZVI and improve Fe... 0 The oxygen molecule activation efficiency. This invention, by comparing nZVI prepared using hexadecyltrimethylammonium bromide (CTAB) as a surfactant, found that structural regulation of nZVI using tetrahydrofuran not only prevents rapid oxidation of nZVI but also enhances the electron contribution rate of nZVI, making Fe... 0 Electrons are transferred to O2 to generate reactive oxygen species (H2O2, ·O2). ‑ , ·OH), thereby improving the oxygen activation efficiency of nZVI.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for synthesizing nano-zero-valent iron using tetrahydrofuran as a reaction medium to improve oxygen activation efficiency and its application. Background Technology

[0002] Typically, tumor treatment often involves the introduction of external energy, such as photodynamic therapy (PDT) and magnetothermal therapy (MHT), which increases the complexity of the procedure and the severity of the treatment conditions. In recent years, research has found that nano-zero-valent iron (nZVI) possesses strong reducing properties (E... θ (Fe 2+ / Fe 0 (nZVI = -0.44V) is widely used as an electron donor in the treatment of organic pollutants in water. Specifically, using nZVI as an electron donor, molecular oxygen can be activated via a two-electron transfer pathway to produce H₂O₂ and Fe. 2+ (Equation 1). Simultaneously, molecular oxygen reacts with the in-situ generated Fe... 2+ The reaction generates O2 - (Equation 2), with highly active ·O2 - Continue with Fe 2+ The reaction produces H2O2 (Equation 3), which can be further reduced to highly reactive ·OH (Equation 4). However, under normal circumstances, nZVI is prone to aggregation and has a limited electron contribution rate, resulting in a very limited ROS yield, which seriously hinders the application of nZVI in the field of tumor therapy.

[0003] O2+Fe 0 +2H + →Fe 2+ +H2O2 (1)

[0004] O2+Fe 2+ →·O2 - +Fe 3+ (2)

[0005] ·O2 - +2H + +Fe 2+ →H₂O₂ + Fe 3+ (3)

[0006] H2O2+e - →·OH+OH - (4)

[0007] As is well known, the synthesis and preparation of nZVI has long been a research hotspot in the field of nanotechnology. Traditionally, the synthesis and preparation of nZVI have often been limited by complex operating procedures, unstable product properties, uncontrollable nanoparticle morphology, and Fe... 0 Problems include low content. For example, mechanical ball milling consumes a lot of energy and the specialized equipment is expensive; the thermal decomposition of organometallic precursors is unstable and often contains organic impurities. Although the classic liquid-phase reduction method has advantages such as simple operating conditions and controllable product morphology, the prepared nZVI is easily oxidized by oxygen and has disadvantages such as low electron contribution rate. Currently, research has found that some organic compounds can react with Fe. 2+ Coordination complexation modulates the morphology of nZVI nanoparticles during synthesis and preparation, resulting in uniform dispersion and a smaller size (approximately 200 nm). Based on this, we utilize a liquid-phase reduction method to introduce tetrahydrofuran (THF) and Fe into the synthesis of nZVI. 2+ Coordination was performed to prepare nZVI-THF with controllable morphology. Meanwhile, we compared it with nZVI-CTAB synthesized using hexadecyltrimethylammonium bromide (CTAB) as a surfactant, and found that nZVI-THF synthesized using THF as the reaction medium had a higher electron contribution rate.

[0008] Given that nZVI-THF prepared by liquid-phase reduction has the characteristics of uniform nanoparticle dispersion, small particle size, high ROS yield, and good biocompatibility, we developed nZVI-THF-ss-SiO2 cancer therapy nanomaterials. Using nZVI-CTAB-ss-SiO2 as a control material, we highlighted the ROS generation capacity of nZVI-THF and the tumor cell killing effect of nZVI-THF-ss-SiO2.

[0009] Taking the above considerations into account, this invention directly introduces THF for coordination reduction during the synthesis of nZVI, and it has been found that THF can effectively reduce Fe during the synthesis process. 0 This reduces losses and enhances the electron contribution rate of nZVI, making Fe... 0 It more efficiently promotes the activation of molecular oxygen (Equations 1-4) and promotes ROS generation. On the other hand, nZVI with surface modification using CTAB cannot effectively protect Fe. 0 It produces only trace amounts of ROS. Furthermore, cell experiments showed that the nZVI-THF-ss-SiO2 material, encapsulated with disulfide-bonded SiO2, exhibits excellent ROS production capacity in tumor cells (HpG2 cells), suggesting that the nZVI-THF-ss-SiO2 material can be applied to tumor therapy.

[0010] The inventors conducted the following searches regarding the relevant content of this application:

[0011] 1. Search results from http: / / scholar.glgoo.com / (September 22, 2024)

[0012] Search keywords Search results Patented tetrahydrofuran enhances the oxygen activation efficiency of nZVI All four items are irrelevant to this method. Patented tetrahydrofuran enhances the electron contribution rate of nZVI One item is irrelevant to this method. Patented tetrahydrofuran synthesis of nZVI All 7 items are irrelevant to this method. <![CDATA[Patent Tetrahydrofuran Improves Fe Content in nZVI 0 Content]]> All five items are irrelevant to this method.

[0013] 2. Search results from CNKI (China National Knowledge Infrastructure): (September 22, 2024)

[0014] Search Method 1:

[0015] Title: Tetrahydrofuran Improves Oxygen Activation Efficiency of nZVI (0 items)

[0016] Title: Tetrahydrofuran-enhanced nZVI electron contribution rate: 0 terms.

[0017] The article titled "Synthesis of tetrahydrofuran nZVI257" is irrelevant to this method.

[0018] Title: Tetrahydrofuran Increases Fe in nZVI 0 Content of 0 items.

[0019] Search Method Two:

[0020] The full text states that tetrahydrofuran enhances the oxygen activation efficiency of nZVI in 336 ways, none of which are relevant to this method.

[0021] The full text of the 1210 electron contribution terms of tetrahydrofuran-enhanced nZVI is irrelevant to this method.

[0022] The entire text—items related to the synthesis of tetrahydrofuran (nZVI123469)—are irrelevant to this method.

[0023] Full text-----Tetrahydrofuran increases Fe in nZVI 0 The content of 916 items was not related to this method.

[0024] Search Method 3:

[0025] Keywords: Tetrahydrofuran (item 2297), none of which are relevant to this method.

[0026] Keywords: Oxygen activation (item 141), none of which are relevant to this method.

[0027] Keywords: Nano-zero valent iron (1388 items), none of which are relevant to this method. Summary of the Invention

[0028] Therefore, this invention provides a method for synthesizing nano-zero-valent iron (nZVI) using tetrahydrofuran as a reaction medium to improve oxygen activation efficiency and its application. The strong reducing properties of nano-zero-valent iron (nZVI) endow it with the ability to act as an electron donor to activate molecular oxygen and generate a large amount of ROS. However, conventional nZVI preparation methods cannot effectively guarantee a high electron contribution rate and uniform dispersion with small size, especially regarding the Fe on the nZVI surface during the preparation process. 0 The surface of nZVI is easily oxidized by O2 in the environment, resulting in low oxygen molecule activation efficiency and low ROS generation in subsequent applications. This invention highlights the feasibility of the nZVI-THF synthesis method and the higher oxygen activation efficiency of nZVI-THF by comparing nZVI-CTAB synthesized with hexadecyltrimethylammonium bromide (CTAB) as a surfactant and nZVI-THF synthesized with THF as a reaction medium.

[0029] This invention relates to a method for synthesizing nano-zero-valent iron using tetrahydrofuran as a reaction medium to improve oxygen activation efficiency, comprising the following steps:

[0030] Under N2 atmosphere, 20 mL of tetrahydrofuran was added to a conical flask (reactor); 14 mg of ferrous sulfate heptahydrate was weighed, dissolved in 4 mL of dilute sulfuric acid solution (pH=3), and then added to the conical flask. The mixture was ultrasonically dispersed until it was uniformly dispersed, and the solution in the flask changed from clear to yellow; 0.1 g of sodium borohydride was weighed, dissolved in 1 mL of dilute sulfuric acid solution (pH=3), and then added dropwise to the conical flask. The reaction solution in the flask gradually changed from yellow to black. The mixture was ultrasonically dispersed for 80 min. The black solution in the conical flask was removed, centrifuged immediately, and the precipitate was collected. The precipitate was washed with deionized water (for deoxygenation) and anhydrous ethanol, and then vacuum dried at room temperature to obtain black nZVI-THF solid powder.

[0031] The nano-zero-valent iron material prepared by this invention has a size of about 200 nm.

[0032] The application of the nano-zero-valent iron material prepared by this invention in the preparation of tumor therapeutic drug formulations.

[0033] This invention employs SiO2 containing disulfide bonds to encapsulate nZVI-THF material, thereby improving the biocompatibility of nZVI-THF material and the specific response of cancer cells to the material in subsequent tumor treatment. Through optimization experiments, this invention ultimately determined that the above parameters ensure a silicon dioxide outer layer of suitable thickness after encapsulation.

[0034] Specifically, the steps include the following:

[0035] First, weigh 5 mg of nZVI-THF material and DSPE-mPEG. 2000Add 5 mg to a glass bottle, add 6.35 mL of anhydrous ethanol to the glass bottle, and sonicate for 1 min to disperse evenly.

[0036] Then, add 1.35 mL of deionized water and 400 μL of ammonia (5.38 mol / L) to the glass bottle and sonicate for 1 min to disperse evenly.

[0037] Next, 3.4 μL of tetraethyl silicate and 1.6 μL of bis-[3-(triethoxysilyl)propyl]-disulfide were added to a glass bottle and sonicated for 5 min. Then, 0.1 μL of bis-[3-(triethoxysilyl)propyl]-tetrasulfide was added and sonicated for 2 h.

[0038] Finally, after the reaction was complete, the precipitate was centrifuged and collected, washed with anhydrous ethanol, and dried under vacuum at room temperature to obtain the nZVI-THF-ss-SiO2 material.

[0039] The nZVI-THF-ss-SiO2 material of this invention can induce Fe in the tumor microenvironment. 0 It promotes the activation of molecular oxygen, resulting in high levels of ROS within tumor cells.

[0040] The reason why the nZVI-THF material prepared by this invention using THF as the reaction medium has good ROS generation ability is that THF mainly enhances the oxygen activation efficiency of nZVI through the following pathways:

[0041] Approach: Introducing THF during the preparation of nZVI materials can significantly increase the total number of electrons provided by the nZVI-THF material, thereby activating more molecular oxygen on the surface of the nZVI-THF material and generating more ROS.

[0042] The nZVI-THF material has a high electron contribution rate and excellent ROS generation ability. At the same time, the nZVI-THF-ss-SiO2 encapsulated with disulfide bonds can induce oxygen activation in cells to generate a large amount of reactive oxygen species, induce the accumulation of lipid peroxides in cells, and effectively promote ferroptosis in tumor cells.

[0043] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0044] 1. This invention uses a liquid-phase reduction method with tetrahydrofuran as the reaction medium to synthesize nZVI materials, which effectively enhances the electron contribution rate of nZVI materials and improves the oxygen molecule activation efficiency on the surface of nZVI materials, providing a new approach to improve the oxygen activation efficiency of nZVI materials.

[0045] 2. The nZVI-THF-ss-SiO2 material described in this invention has the potential to act as a highly efficient ROS inducer in the tumor microenvironment. Attached Figure Description

[0046] Figure 1 These are transmission electron microscope (TEM) images of nZVI-THF and nZVI-CTAB materials. Figure 1 It can be seen that nZVI-THF has a spherical morphology with a single nanoparticle size of about 200 nm; nZVI-CTAB has a spherical morphology with a single nanoparticle size of about 200 nm.

[0047] Figure 2 This is the X-ray photoelectron spectroscopy (XPS) of nZVI-THF and nZVI-CTAB materials. From... Figure 2 It can be seen that the surface of ZVI-THF material has a high Fe content. 0 The content, while Fe is difficult to detect on the nZVI-CTAB surface. 0 This result indicates that Fe on the surface of ZVI-THF material 0 The Fe content is much higher than that on the surface of nZVI-CTAB material. 0 content.

[0048] Figure 3 This is a graph showing the experimental results of the electron contribution rate of nZVI-THF and nZVI-CTAB materials measured using a gas chromatography-thermal conductivity detector (GC-TCD) system. From... Figure 3 It can be seen that the hydrogen production of the nZVI-THF material is significantly higher than that of the nZVI-CTAB material, which indicates that the electron contribution rate of the nZVI-THF material is higher than that of the nZVI-CTAB material.

[0049] Figure 4 The UV-Vis absorption spectra are obtained by detecting the difference in ROS levels between nZVI-THF and nZVI-CTAB materials using methylene blue (MB). Figure 4 The results showed that the absorbance of MB detected in the nZVI-CTAB material experimental group decreased less, while the absorbance of MB detected in the nZVI-THF material experimental group decreased significantly under the same conditions. This indicates that the nZVI-THF material can more effectively activate molecular oxygen and generate more ROS.

[0050] Figure 5 The fluorescence spectrum of ROS generated by oxygen activation in nZVI-THF material was detected using DCFH-DA (2,7-dichlorofluorescein diacetate) as a probe. Figure 5It can be seen that, compared with nZVI-CTAB, the fluorescence emission intensity of DCFH-DA detected in the nZVI-THF material experimental group is significantly improved at 530 nm, indicating that nZVI synthesized using THF can improve the oxygen activation efficiency of nZVI and generate more ROS.

[0051] Figure 6 This figure shows the experimental results of further evaluating the oxygen activation ability of nZVI-THF materials using TPA (terephthalic acid) as an indicator. In the presence of ·OH, TPA undergoes an addition reaction, forming hTPA (hydroxyterephthalate) intramolecularly. hTPA is more prone to fluorescence resonance energy transfer than TPA, thus enhancing the fluorescence signal intensity. From... Figure 6 It can be seen that the fluorescence signal intensity of the nZVI-THF material experimental group is the highest, approximately twice that of the nZVI-CTAB material experimental group. This indicates that nZVI synthesized using THF can improve the oxygen activation efficiency of nZVI, generating more ·OH.

[0052] Figure 7 This figure shows the experimental results of further evaluating the oxygen activation ability of nZVI-THF materials using DHR 123 (dihydrorhodamine 123) as an indicator. The DHR 123 molecule contains a dichlorofluorescein group and a diacetate group. When DHR 123 reacts with ·O2... - During the reaction, ·O2 - It is reduced to the dichlorofluorescein group of DHR 123, changing it from a non-fluorescent state to a fluorescent state. From Figure 7 It is clearly evident that the fluorescence signal intensity of the nZVI-THF material experimental group was the highest, approximately three times that of the nZVI-CTAB material experimental group. This indicates that the nZVI-THF material can improve the molecular oxygen activation efficiency, generating more ·O2. - .

[0053] Figure 8 This is a graph showing the experimental results of further exploring the oxygen activation pathway of nZVI-THF materials using titanium sulfate (Ti(SO4)2) as an indicator. From... Figure 8 It is clear that the fluorescence signal intensity of the nZVI-THF material experimental group is the highest, approximately 11 times that of the nZVI-CTAB material experimental group. This indicates that the nZVI-THF material can improve the molecular oxygen activation efficiency and generate more H2O2.

[0054] Figure 9 This is a graph showing the experimental results of further exploring the oxygen activation pathway of nZVI-THF materials using DHR 123 (dihydrorhodamine 123) as an indicator. From... Figure 9It is clearly evident that the fluorescence signal intensity of the nZVI-THF material is significantly enhanced under oxygen-rich conditions compared to air conditions. This indicates the presence of ·O2, a molecular oxygen activation product, in the nZVI-THF material. - This further illustrates that the pathway for ROS generation in nZVI-THF materials is oxygen molecule activation.

[0055] Figure 10 This is a graph showing the experimental results obtained using the free radical scavenger DMPO for EPR detection. From... Figure 10 As can be seen, both the nZVI-THF and nZVI-CTAB material experimental groups exhibit a clear 1:2:2:1 EPR signal of DMPO-·OH, indicating the presence of ·OH as the molecular oxygen activation product in both materials. Simultaneously, DMPO-·O2 was detected in the nZVI-THF material experimental group. - The six-peak EPR signal was observed, while the nZVI-CTAB material experimental group had difficulty detecting DMPO-·O2. - The sextet EPR signal indicates the presence of ·O2, a molecular oxygen activation product, in the nZVI-THF material. - However, the molecular oxygen activation ability of nZVI-CTAB material is limited, making it difficult to detect ·O2 by EPR. - .

[0056] Figure 11 This image shows the experimental results obtained by monitoring intracellular ROS levels using confocal laser scanning microscopy with DCFH-DA as a fluorescent probe. Figure 11 It can be clearly observed that, compared with nZVI-CTAB-ss-SiO2, the HpG2 cells (human liver cancer cells) treated with nZVI-THF-ss-SiO2 material showed obvious green fluorescence, indicating that nZVI-THF-ss-SiO2 can induce intracellular oxygen activation and generate more ROS.

[0057] Figure 12 These are transmission electron microscope (TEM) images of nZVI-THF-ss-SiO2 material after encapsulation with disulfide-bonded SiO2. Figure 12 As can be seen, nZVI-THF has a spherical morphology, with individual nanoparticles having a size of about 200 nm, and the nanoparticles have a distinct encapsulation layer on the outside. Detailed Implementation

[0058] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.

[0059] Example 1: Preparation of the target product

[0060] 1. Preparation of nZVI-THF and nZVI-CTAB materials

[0061] Preparation of nZVI-THF material: Under N2 atmosphere, 20 mL of tetrahydrofuran was added to a conical flask (reactor); 14 mg of ferrous sulfate heptahydrate was weighed, dissolved in 4 mL of dilute sulfuric acid solution (pH=3), and then added to the conical flask. The mixture was ultrasonically dispersed until it was uniformly dispersed. The solution in the flask changed from clear to yellow. 0.1 g of sodium borohydride was weighed, dissolved in 1 mL of dilute sulfuric acid solution (pH=3), and then added dropwise to the conical flask. The reaction solution in the flask gradually changed from yellow to black. The mixture was ultrasonically dispersed for 80 min. The black solution in the conical flask was removed, centrifuged immediately, and the precipitate was collected. The precipitate was washed with deionized water (for deoxygenation) and anhydrous ethanol, and then vacuum dried at room temperature to obtain black nZVI-THF solid powder.

[0062] Preparation of nZVI-CTAB material: Under N2 atmosphere, 20 mL of dilute sulfuric acid solution (pH=3) was added to a conical flask (reactor); 14 mg of ferrous sulfate heptahydrate and 52.3 mg of CTAB were weighed, dissolved in 4 mL of dilute sulfuric acid solution (pH=3), and then added to the conical flask. The mixture was ultrasonically dispersed until clear; 0.1 g of sodium borohydride was weighed, dissolved in 1 mL of dilute sulfuric acid solution (pH=3), and then added dropwise to the conical flask. The reaction solution gradually changed from clear to black and produced a large amount of foam. The mixture was ultrasonically dispersed for 80 min. The black solution in the conical flask was removed, centrifuged immediately, and the precipitate was collected. The precipitate was washed with deionized water (for deoxygenation) and anhydrous ethanol, and then vacuum dried at room temperature to obtain black nZVI-CTAB solid powder.

[0063] 2. Preparation of nZVI-THF-ss-SiO2 and nZVI-CTAB-ss-SiO2 materials

[0064] Preparation of nZVI-THF-ss-SiO2 material: First, weigh 5 mg of nZVI-THF material and 5 mg of DSPE-mPEG. 2000 Add to a glass bottle, add 6.35 mL of anhydrous ethanol, and sonicate for 1 min to disperse evenly.

[0065] Then, add 1.35 mL of deionized water and 400 μL of ammonia (5.38 mol / L) to the glass bottle and sonicate for 1 min to disperse evenly.

[0066] Next, 3.4 μL of tetraethyl silicate and 1.6 μL of bis-[3-(triethoxysilyl)propyl]-disulfide were added to a glass bottle and sonicated for 5 min. Then, 0.1 μL of bis-[3-(triethoxysilyl)propyl]-tetrasulfide was added and sonicated for 2 h.

[0067] Finally, after the reaction was complete, the precipitate was centrifuged and collected, washed with anhydrous ethanol, and dried under vacuum at room temperature to obtain nZVI@THF-ss-SiO2 material.

[0068] Preparation of nZVI-CTAB-ss-SiO2 material: First, weigh 5 mg of nZVI-CTAB material and 5 mg of DSPE-mPEG. 2000 Add to a glass bottle, add 6.35 mL of anhydrous ethanol, and sonicate for 1 min to disperse evenly.

[0069] Then, add 1.35 mL of deionized water and 400 μL of ammonia (5.38 mol / L) to the glass bottle and sonicate for 1 min to disperse evenly.

[0070] Next, 3.4 μL of tetraethyl silicate and 1.6 μL of bis-[3-(triethoxysilyl)propyl]-disulfide were added to a glass bottle and sonicated for 5 min. Then, 0.1 μL of bis-[3-(triethoxysilyl)propyl]-tetrasulfide was added and sonicated for 2 h.

[0071] Finally, after the reaction was complete, the precipitate was centrifuged and collected, washed with anhydrous ethanol, and dried under vacuum at room temperature to obtain the nZVI-CTAB-ss-SiO2 material.

[0072] Example 2: Investigation on the effect of using THF as a reaction medium on the enhancement of the electron contribution rate of nZVI

[0073] Electron contribution rate determination. First, accurately measure the volume of the glass bottle, add 10 mg of sample (nZVI-THF or nZVI-CTAB) to the bottle, gently tilt it to one side, and then introduce N2 into the other side of the bottom of the bottle for 10 min. Immediately seal the bottle opening with a rubber stopper. Next, add 15 mL of 2.4 mol / L HCl solution while avoiding contact with any particles. Sonicate for 2 min to ensure sufficient contact, and let stand for 2 h to allow complete reaction. Finally, calculate the nZVI electron contribution rate for each sample by measuring H2 in the headspace using a gas chromatography-thermal conductivity detector (GC-TCD) system and carrier gas (argon). Triple-parallel experiments were performed for both nZVI-THF and nZVI-CTAB materials.

[0074] Example 3: Improving the oxygen activation efficiency of nZVI using THF as a reaction medium and investigating the oxygen activation products of nZVI-THF materials.

[0075] 1. MB degradation detection of ROS generation

[0076] nZVI-THF material (200 μg / mL) and nZVI-CTAB material (200 μg / mL) were dispersed in MB solution (6 μg / mL), respectively. After sonication for 30 s and reaction for 30 min, the supernatant was collected by centrifugation (10000 rpm, 1 min) and the absorbance change at around 660 nm was recorded by UV-Vis spectroscopy to investigate the degradation difference between nZVI-THF material and nZVI-CTAB material in MB solution.

[0077] 2. ROS probe DCFH-DA detects ROS generation.

[0078] First, prepare DCFH-DA (2mM, DMSO) solution and NaOH (0.01M) solution. Add 100µL DCFH-DA solution and 0.8mL NaOH solution to a 2mL centrifuge tube, mix well, and activate at room temperature in the dark for 30min to prepare the ROS detection solution.

[0079] Next, the nZVI-THF material (200 μg / mL) and the nZVI-CTAB material (200 μg / mL) were dispersed separately in ROS detection solution (200 μL) and sonicated for 30 s to ensure sufficient contact. Under complete darkness, O2 was bubbled into both reaction solutions for 15 min, followed by centrifugation (10000 rpm, 1 min). The supernatant was then collected, and the fluorescence intensity changes around 530 nm were recorded.

[0080] 3. Detection of ·OH generation using the ·OH probe TPA

[0081] First, prepare a TPA (0.5 mM) solution and a NaOH (0.2 mM) solution, mix them thoroughly, and activate them at room temperature in the dark for 30 minutes to prepare the ·OH detection solution.

[0082] Next, the nZVI-THF material (200 μg / mL) and the nZVI-CTAB material (200 μg / mL) were dispersed in OH detection solution (200 μL) and sonicated for 30 s to ensure sufficient contact. Under complete darkness, O2 was bubbled into both reaction solutions for 15 min, and then centrifuged (10000 rpm, 1 min). The supernatant was collected and the fluorescence intensity changes around 425 nm were recorded.

[0083] 4. O2 - DHR 123 probe for O2 detection - generate

[0084] First, prepare a DHR 123 (1 mM) solution, and mix thoroughly by shaking at room temperature in the dark to form a homogeneous solution. - Test solution.

[0085] Next, the nZVI-THF material (200 μg / mL) and the nZVI-CTAB material (200 μg / mL) were dispersed separately in O2. - In the detection solution (20 μL), sonicate for 30 s to ensure full contact. Under complete darkness, purge O2 into both reaction solutions for 15 min, then centrifuge (10000 rpm, 1 min), collect the supernatant, and record the fluorescence intensity change around 525 nm.

[0086] 5. H2O2 Detection Reagent Ti(SO4)2 for Detecting H2O2 Formation

[0087] H₂O₂ concentration was determined using the titanium sulfate (Ti(SO₄)₂) reagent method. Titanium sulfate reagent (4 mg / mL) was prepared by dissolving titanium sulfate in sulfuric acid (ω(H₂SO₄) = 25%). Then, 1.2 mL of nZVI-THF or nZVI-CTAB (50 mg / mL, pH = 6.0) was added dropwise to 2 mL of titanium sulfate reagent at intervals of 2 min, 4 min, and 6 min. After adding nZVI-THF or nZVI-CTAB (50 mg / mL, pH = 6.0), the mixture was allowed to stand for 6 h. The concentration was determined using UV-Vis spectroscopy (λ). Abs The absorption spectrum of the yellow complex (H2[TiO2(SO4)2]) was measured at 415 nm.

[0088] 6. O2 - Probe DHR 123 investigates the oxygen activation pathway of nZVI-THF materials

[0089] Two nZVI-THF material reaction solutions (200 μg / mL) were prepared, and DHR123 solution (10 μL / mL) was added to each solution. Under the complete dark protection, air or O2 was introduced into each of the two reaction solutions for 15 min. After centrifugation (10000 rpm, 1 min), the supernatant was collected and the fluorescence intensity change at around 525 nm was recorded to investigate the electron transfer pathway during the oxygen activation process of nZVI-THF material.

[0090] 7. Electron Spin Resonance Spectroscopy (EPR) Detection of OH

[0091] Using DMPO (5,5-dimethyl-1-pyrroline-N-oxide) as a trapping agent, EPR analysis determined that the ROS generated by oxygen activation of nZVI-THF materials were ·OH and ·O2. - The ROS generated by oxygen activation of nZVI-CTAB material is ·OH, while ·O2 - Difficult to detect. This indicates the presence of molecular oxygen activation products (·O2) in the nZVI-THF material. - The ·O2 generated by the molecular oxygen activation of nZVI-CTAB material -Production is very limited.

[0092] Example 4: Detection of intracellular ROS

[0093] The intracellular ROS production under different treatment conditions was detected using DCFH-DA. HepG2 cells (37℃, 5% CO2) were cultured for 24 h and then co-incubated for 16 h with nZVI-THF-ss-SiO2 (300 μg / mL) and nZVI@CTAB-ss-SiO2 (300 μg / mL), respectively, followed by three washes with PBS. Then, DCFH-DA was added and incubated for 15 min, followed by three more washes with PBS. 1 mL of PBS was added, and fluorescence imaging was observed using a confocal laser scanning microscope.

Claims

1. A method for synthesizing nZVI-THF materials by using tetrahydrofuran as a reaction medium to improve the activation efficiency of nano-zero ferrite, characterized in that: By introducing tetrahydrofuran into the preparation process of nano-zero-valent iron, the morphology of nano-zero-valent iron and the Fe content can be controlled by tetrahydrofuran. 0 The activation efficiency of oxygen molecules is determined by the following steps: Under a nitrogen atmosphere, tetrahydrofuran was added to the reactor; ferrous sulfate heptahydrate was weighed, dissolved completely in a dilute sulfuric acid solution with pH=3, and then added to an Erlenmeyer flask. The solution was ultrasonically dispersed until it changed from clear to yellow; sodium borohydride was weighed, dissolved completely in a dilute sulfuric acid solution, and then added dropwise to the Erlenmeyer flask. The reaction solution in the flask gradually changed from yellow to black. Ultrasonic treatment was continued, and the black solution in the Erlenmeyer flask was removed. The solution was immediately centrifuged and the precipitate was collected. The precipitate was washed with deionized water and anhydrous ethanol and dried under vacuum at room temperature to obtain a black nZVI-THF solid powder.

2. The application of the nZVI-THF material prepared according to claim 1 in the preparation of tumor therapeutic drug formulations, characterized in that: The tumor is liver cancer.

3. The application of the nZVI-THF material prepared according to claim 1 in the preparation of ROS inducers.

4. The application according to claim 2, characterized in that: Encapsulating nZVI-THF materials with SiO2 containing disulfide bonds improves the biocompatibility of nZVI-THF materials and the specific response of cancer cells to the materials in subsequent tumor treatment.

5. The application according to claim 4, characterized in that... Includes the following steps: Step 1: Weigh out nZVI-THF material and DSPE-mPEG 2000 Add to a glass bottle, add anhydrous ethanol to the glass bottle, and ultrasonically disperse until uniform; Step 2: Add deionized water and ammonia to the glass bottle and disperse evenly using ultrasonication; Step 3: Add tetraethyl silicate and bis-[3-(triethoxysilyl)propyl]-disulfide to a glass bottle, sonicate for 5 min, add bis-[3-(triethoxysilyl)propyl]-tetrasulfide, and sonicate to react; Step 4: After the reaction is complete, centrifuge and collect the precipitate, wash with anhydrous ethanol, and vacuum dry at room temperature to obtain nZVI-THF-ss-SiO2 material.

6. The application according to claim 5, characterized in that: In step 1, 5 mg of nZVI-THF material and DSPE-mPEG were weighed out. 2000 Add 5 mg to a glass bottle, add 6.35 mL of anhydrous ethanol to the glass bottle, and disperse evenly by ultrasonication.

7. The application according to claim 5, characterized in that: In step 2, 1.35 mL of deionized water and 400 μL of ammonia water are added to the glass bottle and ultrasonically dispersed evenly.

8. The application according to claim 5, characterized in that: In step 3, 3.4 μL of tetraethyl silicate and 1.6 μL of bis-[3-(triethoxysilyl)propyl]-disulfide were added to a glass bottle and sonicated for 5 min. Then, 0.1 μL of bis-[3-(triethoxysilyl)propyl]-tetrasulfide was added and the mixture was sonicated for 2 h.

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