Method for improving oxygen activation efficiency by nanomaterials 0 Method for improving oxygen activation efficiency by nanomaterials

Abstract

The application discloses a kind of Fe 0 Nanometer material is improved oxygen activation efficiency method, by introducing metformin on the surface of Fe 0 , using metformin to adjust the oxygen reduction route of Fe 0 Surface, improve the oxygen activation efficiency of Fe 0 The application uses metformin to carry out surface modification to Fe 0 Nanometer material, not only can prevent Fe 0 Be quickly oxidized, more importantly, can also adjust the oxygen reduction route of Fe 0 Surface, make Fe 0 Will transfer electron to O2 and generate ROS, so as to improve the oxygen activation efficiency of Fe 0 .

Description

Technical Field

[0001] This invention relates to a method for treating Fe using metformin. 0 Nanomaterials are used for surface modification to effectively enhance Fe 0 Methods for determining oxygen activation efficiency. Background Technology

[0002] Metformin, Metformin is a synthetic biguanide drug, best known as a first-line oral hypoglycemic agent for treating type 2 diabetes. Beyond lowering blood sugar, metformin's other clinical applications are attracting increasing attention. For example, metformin has anti-aging, antiviral, and anti-inflammatory effects, can improve lipid metabolism, and reduce the incidence of cardiovascular complications. Notably, metformin can inhibit the oxygen consumption of tumor cells, showing potential application prospects in anti-tumor therapy. This inspires scientists to utilize metformin in combination with other tumor treatments to achieve synergistic effects and thus superior tumor-killing efficacy.

[0003] Inducing tumor cell death by increasing intracellular ROS levels has gradually become an emerging approach to cancer treatment (e.g., chemokinetic therapy (CDT), photodynamic therapy (PDT), etc.). Research has found that nano-zero-valent iron (Fe)... 0 It has strong reducing properties (E) θ (Fe 2+ / Fe 0 (Equation 1) (equation 1) (equation 1) (equation 2) (equation 3) (equation 4) (equation 5 ...6) (equation 7) (equation 8) (equation 9) 2+ The reaction produces ·OH (Equation 2), therefore Fe 0 It shows great promise for applications in ROS-driven cancer therapy. However, Fe... 0 The ROS yield is very low because most of the O2 easily passes through Fe. 0 The four-electron transfer pathway on the surface is reduced to H2O without producing ROS (Equation 3).

[0004] O2+Fe 0 +2H + →Fe 2+ +H2O2 (1)

[0005] Fe 2+ +H₂O₂→Fe 3+ +·OH+OH - (pH<5)(2)

[0006] O2 + 2Fe 0 +4H+ →2Fe 2+ +2H2O (3)

[0007] Therefore, increasing Fe 0 The oxygen activation efficiency, adjusting Fe 0 The oxygen reduction pathway on the surface is particularly important. In previous studies, researchers have discovered that through Fe... 0 Introducing anions (such as Cl) to the surface - NO3 - SO4 2- HPO4 2- ) can be found in Fe 0 An oxide layer with electron transfer function is formed on the surface, which enables the formation of Fe. 0 Modulation of the oxygen reduction pathway on the surface significantly enhances ROS generation. Therefore, researchers believe that the regulation of Fe... 0 Surface modification is a breakthrough for Fe 0 An effective method for addressing low oxygen activation efficiency.

[0008] Taking the above considerations into account, the present invention utilizes metformin to treat Fe 0 Surface modification of nanomaterials revealed that metformin can alter Fe... 0 The oxygen reduction pathway on the surface allows Fe 0 This more efficiently promotes the activation of molecular oxygen (Equations 1 and 2), thus promoting ROS generation. Our cell experiments show that Fe2+ encapsulated with disulfide-bonded SiO2... 0 @met-ss-SiO2 nanomaterials exhibit superior ROS generation capacity within tumor cells (HpG2 cells), suggesting that Fe... 0 @met-ss-SiO2 nanomaterials can be used in cancer treatment.

[0009] The inventors conducted the following searches regarding the relevant content of this application:

[0010] 1. Search results from http: / / scholar.glgoo.com / (November 20, 2023):

[0011] Search keywords Search results <![CDATA[Patent: Metformin Improves the Oxygen Activation Efficiency of Fe 0 > none <![CDATA[Patent Metformin Regulates the Oxygen Activation Pathway of Fe 0 > none <![CDATA[Patent Metformin Promotes the Activation of Molecular Oxygen by Fe 0 > none Patented metformin promotes the generation of reactive oxygen species none <![CDATA[Patented metformin-modified Fe 0 nanoparticles]]> none

[0012] 2. Search results from CNKI (China National Knowledge Infrastructure): (November 20, 2023)

[0013] Search Method 1:

[0014] Title: Metformin Enhances Fe 0 The oxygen activation efficiency is 0.

[0015] Title: Metformin Regulates Fe0 There are 0 oxygen activation pathways.

[0016] Title: Metformin Promotes Fe 0 There are 0 molecular oxygen activation terms.

[0017] Search Method Two:

[0018] Full text-----Metformin enhances Fe 0 The oxygen activation efficiency is 0.

[0019] Full text-----Metformin regulates Fe 0 The seven oxygen activation pathways are all irrelevant to this method.

[0020] Full text-----Metformin promotes Fe 0 The seven molecular oxygen activation parameters are all irrelevant to this method.

[0021] Search Method 3:

[0022] Keywords: Metformin; 9130 items were not relevant to this method.

[0023] Keywords: Oxygen activation efficiency (19 items), none of which are relevant to this method.

[0024] Keywords: Molecular oxygen activation (5379 items), none of which are relevant to this method. Summary of the Invention

[0025] Therefore, the present invention aims to provide a method for modifying Fe with metformin. 0 Methods to improve oxygen activation efficiency using nanomaterials. Nano-sized zero-valent iron (Fe). 0 The strong reducing properties of Fe enable it to act as an electron donor, undergoing molecular oxygen activation to produce large amounts of ROS. However, since the four-electron transfer pathway is Fe... 0 The main reduction pathway of surface O2 involves the direct conversion of O2 to H2O, which is a key reason for the low ROS generation efficiency. Therefore, this invention addresses this issue by using Fe... 0 Metformin is introduced onto the surface to regulate Fe. 0 Surface oxygen reduction pathway to achieve Fe 0 The oxygen activation efficiency is effectively improved.

[0026] This invention utilizes metformin to modify Fe 0 The method for improving oxygen activation efficiency using nanomaterials includes the following steps:

[0027] Step 1: Fe 0 Preparation of nanomaterials

[0028] A 100 mL three-necked flask was selected, and 35 mL of dilute sulfuric acid solution (pH=3) was added. The mixture was stirred mechanically for 10 min under a N2 atmosphere. 19.8 mg of ferrous sulfate heptahydrate and 74 mg of hexadecyltrimethylammonium bromide were weighed out, dissolved thoroughly in 5 mL of dilute sulfuric acid solution (pH=3), and added to the three-necked flask. The mixture was stirred and dispersed for 10 min. 9.8 mg of sodium borohydride was weighed out, dissolved thoroughly in 3 mL of dilute sulfuric acid solution (pH=3), and added dropwise to the three-necked flask. The solution in the flask quickly turned black. After stirring and dispersing for another 5 min, the black solution in the three-necked flask was removed, immediately centrifuged (10000 rpm, 1 min), and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and dried under vacuum at room temperature for 12 h to obtain black Fe. 0 Nanomaterials.

[0029] Step 2: Fe 0 Preparation of @met nanomaterials

[0030] Select a 10mL glass bottle and weigh out 5mg of Fe. 0 Nanomaterials and 50 mg of metformin were added, followed by 10 mL of anhydrous ethanol in a glass bottle. The mixture was ultrasonically dispersed and magnetically stirred at room temperature for 12 h. After the reaction was complete, the mixture was immediately centrifuged (10,000 rpm, 1 min) and the precipitate was collected. The precipitate was then vacuum dried at room temperature for 12 h to obtain black Fe. 0 @met nanomaterials.

[0031] Step 3: Fe 0 Preparation of @met-ss-SiO2 nanomaterials

[0032] 3a. Select a 25mL glass bottle, add 1.35mL of deionized water and 100μL of ammonia (5.38mol / L), mix well, and then add 3.75mL of anhydrous ethanol to the glass bottle to obtain solution 1.

[0033] 3b. Select a 10mL glass bottle and weigh 5mg of Fe. 0 @met nanomaterials and 5mg amine-polyethylene glycol 2000 (DSPE-mPEG2000) were added to the solution, and then 2.5mL of anhydrous ethanol was added to the glass bottle. The solution was ultrasonically dispersed to obtain solution 2.

[0034] 3c. Select a 10mL glass bottle, measure 8μL of tetraethyl orthosilicate (TEOS) and 3.4μL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTSPD) and add them to the bottle. Then add 289μL of anhydrous ethanol to the glass bottle and sonicate to disperse evenly to obtain solution 3.

[0035] 3d. Add solutions 2 and 3 to solution 1 and sonicate for 2 hours. After the reaction, centrifuge (10,000 rpm, 1 min) and collect the precipitate. Wash three times with water and anhydrous ethanol, respectively, and vacuum dry at room temperature for 12 hours to obtain Fe. 0 @met-ss-SiO2 nanomaterials.

[0036] This invention uses SiO2 containing disulfide bonds to react with Fe. 0 The encapsulation of @met nanomaterials aims to improve Fe 0 The biocompatibility of @met nanomaterials and the specific response of cancer cells to the materials in subsequent tumor treatment. This invention, through optimized experiments, ultimately determined that the above parameters can ensure a silica outer layer of suitable thickness after encapsulation.

[0037] Furthermore, in the process of preparing solution 2, the present invention uses an equal amount of Fe 0 Nanomaterials to replace Fe 0 @met nanomaterials, preparation of Fe 0 -ss-SiO2 nanomaterials were compared and investigated.

[0038] In a first aspect, the present invention provides an Fe 0 Nanomaterials, with a size of around 110 nm.

[0039] Secondly, the present invention provides a method to improve Fe 0 The oxygen activation efficiency, through Fe 0 Metformin can be introduced onto the surface of nanomaterials.

[0040] Thirdly, the present invention provides an Fe 0 @met-ss-SiO2 nanomaterials can induce Fe in the tumor microenvironment 0 It promotes the activation of molecular oxygen, resulting in high levels of ROS within tumor cells.

[0041] This invention modifies Fe with metformin 0 The reason why nanomaterials have good ROS generation ability is that metformin mainly enhances Fe through the following two pathways. 0 Oxygen activation efficiency:

[0042] Approach 1: The presence of metformin can prevent Fe 0 It is rapidly oxidized by oxygen in aqueous solution, resulting in more Fe 0 It is retained, resulting in more oxygen-activated products.

[0043] Pathway 2: The presence of metformin can significantly inhibit Fe 0 Metformin undergoes a four-electron transfer pathway with O2. Metformin regulates Fe...0 The oxygen reduction pathway on the surface allows Fe 0 It is easier to transfer electrons to O2 to generate more ROS.

[0044] Fe 0 @met nanomaterials have high Fe content 0 The content and better ROS generation ability, and Fe 0 @met nanomaterials, after being encapsulated with SiO2 containing 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.

[0045] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0046] 1. This invention utilizes metformin to treat Fe 0 Surface modification with nanomaterials not only effectively preserves Fe 0 The content of Fe was also achieved. 0 Regulation of the oxygen reduction pathway on the surface is crucial for improving Fe... 0 This provides a new approach to understanding oxygen activation efficiency.

[0047] 2. The Fe described in this invention 0 @met-ss-SiO2 nanomaterials have the potential to serve as highly efficient ROS inducers in the tumor microenvironment. Attached Figure Description

[0048] Figure 1 It is Fe 0 @met element mapping diagram of nanomaterials. Figure 1 The results show that the elements (O, N, and Fe) in the nanomaterial are uniformly distributed, proving that metformin successfully modified Fe. 0 Nanomaterials.

[0049] Figure 2 It is a precursor Fe 0 Nanomaterials, end product Fe 0 @met nanomaterials, and Fourier transform infrared (FTIR) spectroscopy of metformin. Because metformin at 3297 cm⁻¹... -1 and 3372cm -1 (NH-stretched), 1627cm -1 and 1574cm -1 (CN-Stretch), 3174cm -1 (CH-stretch) and 934cm -1 There is an absorption peak at (CH- deformation). Figure 2 Fe 0The FTIR of @met nanomaterials shows that at 3174 cm⁻¹ -1 1627cm -1 and 1574cm -1 The presence of a distinct absorption peak nearby proves that metformin successfully modified Fe. 0 Nanomaterials.

[0050] Figure 3 It is a precursor Fe 0 Nanomaterials, end product Fe 0 X-ray photoelectron spectroscopy (XPS) of @met nanomaterials. From Figure 3 It can be seen that Fe 0 Fe on the surface of nanomaterials 0 The content is almost zero. And in Fe... 0 @met nanomaterials can exhibit high Fe 2p spectra. 0 The result indicates the content of Fe. 0 @met nanomaterials Fe 0 Content higher than Fe 0 Fe nanomaterials 0 content.

[0051] Figure 4 It is Fe 0 Nanomaterials and Fe 0 The image shows the results of an experiment on the oxidation of @met nanomaterials in water at different times. From... Figure 4 It can be seen that even without the introduction of O2, Fe 0 The aqueous solution of the nanomaterial was significantly oxidized after 1 hour, while Fe... 0 When O2 is bubbled into an aqueous solution of @met nanomaterials, the oxidation tendency is significantly suppressed. After 1 hour, Fe... 0 @met nanomaterials in aqueous solution and Fe isolated from O2 (in N2 atmosphere) 0 The fact that the aqueous solutions of nanomaterials showed no significant color difference indicates that metformin is beneficial in protecting Fe. 0 It is not rapidly oxidized, thus greatly preserving Fe. 0 The content of.

[0052] Figure 5 It uses MB to detect Fe 0 UV-Vis absorption spectrum of ·OH generated by oxygen activation in @met nanomaterials. Figure 5 The results showed that in Fe-containing 0 The absorbance of MB detected in the nanomaterial experimental group showed almost no change, while that in the Fe-containing group... 0The absorbance of MB detected in the @met nanomaterials experimental group decreased significantly after 3 minutes, and the degradation rate of MB was about 60% after 10 minutes, indicating that Fe 0 @met nanomaterials can undergo molecular oxygen activation to produce more ·OH.

[0053] Figure 6 The detection of Fe was performed using DCFH-DA as a probe. 0 Fluorescence spectrum of ROS generated by oxygen activation in @met nanomaterials. Figure 6 It can be seen that in the presence of Fe 0 The DCFH-DA fluorescence emission intensity at 530 nm detected in the @met nanomaterials experimental group was significantly increased, indicating that metformin can promote Fe 0 The oxygen activation efficiency is improved, resulting in the generation of more ROS.

[0054] Figure 7 TMB was used as an indicator to further evaluate Fe. 0 The experimental results of the oxygen activation ability of @met nanomaterials are shown in the figure. TMB, in the presence of ROS, can undergo a catalytic oxidation reaction with ROS to produce a bright blue pigment (OX-TMB). From... Figure 7 It can be seen that Fe 0 @met nanomaterials showed the most obvious blue color development in TMB solutions, especially in solutions containing Fe. 0 The UV-Vis absorption spectrum of the @met nanomaterials experimental group shows a significant enhancement of the characteristic peak of oxidized TMB (OX-TMB) near 650 nm, indicating that metformin can promote Fe 0 The oxygen activation efficiency is improved, resulting in the generation of more ROS.

[0055] Figure 8 The UV-Vis absorption spectra of MB degradation experiments using IPA (isopropanol) as an OH scavenger are used to investigate Fe. 0 Molecular oxygen activation products of @met nanomaterials. From Figure 8 It is clear that the degradation of MB is significantly inhibited in the presence of IPA, indicating that Fe 0 The presence of ·OH, a molecular oxygen activation product in @met nanomaterials, further illustrates that metformin regulates Fe. 0 The oxygen activation pathway.

[0056] Figure 9 The UV-Vis absorption spectra of MB degradation experiments using CAT (catalase) as an H2O2 scavenger were used to investigate Fe. 0 Molecular oxygen activation products of @met nanomaterials. From Figure 9It is clear that the degradation of MB is significantly inhibited in the presence of CAT, indicating that Fe 0 The presence of H2O2, a molecular oxygen activation product of @met nanomaterials, further illustrates that metformin regulates Fe. 0 The oxygen activation pathway.

[0057] 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, in the presence of Fe 0 The @met nanomaterials experimental group exhibited a very strong DMPO-·OH 1:2:2:1 EPR signal, further illustrating the presence of Fe. 0 @met nanomaterials contain molecular oxygen activation products, specifically ·OH.

[0058] 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 seen that in Fe 0 The HpG2 cells (human liver cancer cells) treated with @met-ss-SiO2 nanomaterials showed significant green fluorescence, indicating that Fe 0 @met-ss-SiO2 can induce intracellular oxygen activation, generating more ROS. Detailed Implementation

[0059] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.

[0060] Example 1: Preparation of the target product

[0061] 1. Fe 0 Preparation of nanomaterials

[0062] A 100 mL three-necked flask was selected, and 35 mL of dilute sulfuric acid solution (pH=3) was added. The mixture was stirred mechanically for 10 min under a nitrogen atmosphere. 19.8 mg of ferrous sulfate heptahydrate and 74 mg of hexadecyltrimethylammonium bromide were weighed, dissolved thoroughly in 5 mL of dilute sulfuric acid solution (pH=3), and added to the three-necked flask. The mixture was stirred and dispersed for 10 min. 9.8 mg of sodium borohydride was weighed, dissolved thoroughly in 3 mL of dilute sulfuric acid solution (pH=3), and added dropwise to the three-necked flask. The solution in the flask quickly turned black. After continuing to stir and disperse for 5 min, the black reaction solution in the three-necked flask was removed, immediately centrifuged (10000 rpm, 1 min), and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and dried under vacuum at room temperature for 12 h to obtain black Fe. 0 Nanomaterials.

[0063] 2. Fe 0 Preparation of @met nanomaterials

[0064] Select a 10mL glass bottle and weigh out 5mg of Fe. 0 Nanomaterials and 50 mg of metformin were added, followed by 10 mL of anhydrous ethanol in a glass bottle. The mixture was then ultrasonically dispersed. After magnetically stirring the reaction solution at room temperature for 12 hours, the solution was removed, immediately centrifuged (10,000 rpm, 1 min), and the precipitate was collected. The precipitate was then vacuum dried at room temperature for 12 hours to obtain black Fe. 0 @met nanomaterials.

[0065] 3. Fe 0 Preparation of @met-ss-SiO2 nanomaterials

[0066] Solution 1: Select a 25mL glass bottle, add 1.35mL of deionized water and 100uL of ammonia (5.38mol / L), mix well, and then add 3.75mL of anhydrous ethanol to the glass bottle.

[0067] Solution 2: Select a 10mL glass bottle and weigh 5mg of Fe. 0 @met nanomaterials and 5mg amine-polyethylene glycol 2000 (DSPE-mPEG2000) were added to the mixture, and then 2.5mL of anhydrous ethanol was added to the glass bottle. The mixture was then ultrasonically dispersed until homogeneous.

[0068] Solution 3: Select a 10mL glass bottle, measure 8uL of tetraethyl orthosilicate (TEOS) and 3.4uL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTSPD) and add them to the bottle. Then add 289uL of anhydrous ethanol to the glass bottle and ultrasonically disperse the solution evenly.

[0069] Solution 2 and solution 3 were added to solution 1. The glass bottle containing solution 1 was placed in an ultrasonic machine and sonicated for 2 hours. After the reaction was complete, the solution was transferred to a centrifuge tube, centrifuged (10,000 rpm, 1 min), and the precipitate was collected. The precipitate was washed three times with water and anhydrous ethanol, and dried under vacuum at room temperature for 12 hours to obtain Fe. 0 @met-ss-SiO2 nanomaterials.

[0070] 4. Fe 0 Preparation of -ss-SiO2 nanomaterials

[0071] Fe 0 Preparation method of -ss-SiO2 nanomaterials and Fe 0 The preparation method of @met-ss-SiO2 nanomaterials is similar. Except that during the preparation of solution 2, an equal amount of Fe... 0Nanomaterials to replace Fe 0 @met nanomaterials, other experimental procedures remain unchanged.

[0072] Example 2: Metformin for Fe 0 Investigation on the inhibitory effect of oxidation

[0073] Experimental Group 1: At room temperature, Fe 0 Nanomaterials (3 mg) were uniformly dispersed in 6 mL of deionized water (containing oxygen), and the color change of the solution was observed at regular intervals (0 h, 0.5 h, 1 h).

[0074] Experimental Group 2: At room temperature, Fe 0 Nanomaterials (3 mg) were uniformly dispersed in 6 mL of deionized water (containing oxygen). Nitrogen gas was introduced into the deionized water, and the color change of the solution was observed at regular intervals (0 h, 0.5 h, 1 h).

[0075] Experimental Group 3: At room temperature, Fe 0 Nanomaterials (3 mg) were uniformly dispersed in 6 mL of deionized water (containing oxygen). Oxygen was introduced into the deionized water, and the color change of the solution was observed at regular intervals (0 h, 0.5 h, 1 h).

[0076] Experimental Group 4: At room temperature, Fe 0 @met nanomaterials (3mg) were uniformly dispersed in 6mL of deionized water (containing oxygen), and then oxygen was injected into the deionized water for 10min. The color change of the solution was observed at time intervals (0h, 0.5h, 1h).

[0077] Example 3: Metformin enhances Fe 0 The oxygen activation efficiency of Fe was investigated. 0 Oxygen activation products of @met nanomaterials

[0078] 1. MB degradation detection and OH generation

[0079] Experimental group 1: Fe 0 Nanomaterials (200 μg / mL) and Fe 0 @met nanomaterials (200 μg / mL) were dispersed in MB (5 μg / mL) solution. After reacting for 3 min, the mixture was centrifuged (10000 rpm, 1 min), and the supernatant was collected. The absorbance change around 660 nm was recorded using UV-Vis spectroscopy to investigate the effect of metformin on Fe. 0 The degradation effect of the modified solution on MB solution.

[0080] Experimental group 2: Fe 0 Nanomaterials (200 μg / mL) and Fe 0@met nanomaterials (200 μg / mL) were dispersed in MB (5 μg / mL) solution. After reacting for 10 min, the mixture was centrifuged (10000 rpm, 1 min), and the supernatant was collected. The absorbance change around 660 nm was recorded using UV-Vis spectroscopy to investigate the effect of metformin on Fe. 0 The degradation effect of the modified solution on MB solution.

[0081] To further investigate the regulation of Fe by metformin 0 The oxygen reduction pathway on the surface was investigated by introducing the ROS scavengers isopropanol (IPA, 10 μg / mL) and catalase (CTA, 10 μg / mL) based on the above experiments. The type of ROS generated was determined by observing the degradation of MB.

[0082] 2. ROS probe DCFH-DA detects ROS generation.

[0083] Prepare DCFH-DA (2mM, DMSO) solution and NaOH (0.01M) solution. Select a suitable reaction vessel, add 100 μL of DCFH-DA solution and 0.8 mL of NaOH solution, mix thoroughly, and activate at room temperature in the dark. After 30 min, dilute the mixture with 3.1 mL of PBS solution. Prepare an identical solution following the same procedure.

[0084] Finally, add 2 mL of Fe to one portion of the solution. 0 Nanomaterials (1.25 mg / mL), another portion containing Fe 0 After mixing the @met nanomaterial (1.25 mg / mL) solution thoroughly, centrifuge (10000 rpm, 1 min) for 10 min, and record the fluorescence intensity change at around 530 nm.

[0085] 3. TMB oxidation colorimetric assay for the generation of ·OH

[0086] Fe 0 Nanomaterials (200 μg / mL) and Fe 0 @met nanomaterials (200 μg / mL) were uniformly dispersed in TMB (40 μg / mL) solution. After 1 h, the mixture was centrifuged (10000 rpm, 1 min), and the supernatant was collected to measure its UV-Vis absorption spectrum.

[0087] 4. Electron Spin Resonance Spectroscopy (ESR) Detection of OH

[0088] Using DMPO as a scavenger, the generated ROS was determined to be ·OH by ESR (DMPO: 5,5-Dimethyl-1-pyrroline N-oxide, a free radical scavenger; EPR: electron spin resonance spectroscopy), indicating that metformin can promote Fe 0 The oxygen activation efficiency is increased, resulting in the production of more ·OH.

[0089] Example 4: Detection of intracellular ROS

[0090] 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 treated with metformin (15 μg / mL), Fe... 0 -ss-SiO2 nanomaterials (85 μg / mL) and Fe 0 @met-ss-SiO2 nanomaterials (100 μg / mL) were co-incubated for 2 h, followed by washing three times with PBS. Then, DCFH-DA was added and incubated for 15 min, followed by washing three more times with PBS. 1 mL of PBS was added, and fluorescence imaging was observed using a confocal laser scanning microscope.

Claims

1. Modification of Fe using metformin 0 A method for improving oxygen activation efficiency using nanomaterials, characterized by: Through Fe 0 Metformin is introduced onto the surface to regulate Fe. 0 Surface oxygen reduction pathway, increasing Fe 0 oxygen activation efficiency; Includes the following steps: Step 1: Fe 0 Preparation of nanomaterials Under a nitrogen atmosphere, a dilute sulfuric acid solution with a pH of 3 was added to the reactor. 19.8 mg of ferrous sulfate heptahydrate and 74 mg of hexadecyltrimethylammonium bromide were weighed, dissolved thoroughly in the dilute sulfuric acid solution, and added to the reactor, where they were stirred and dispersed evenly. 9.8 mg of sodium borohydride was weighed, dissolved thoroughly in the dilute sulfuric acid solution, and added dropwise to a three-necked flask. The solution in the flask quickly turned black. The mixture was stirred and dispersed for another 5 minutes. The black solution in the three-necked flask was removed, immediately centrifuged, and the precipitate was collected. The precipitate was washed with anhydrous ethanol and dried under vacuum at room temperature to obtain black Fe. 0 Nanomaterials; Step 2: Fe 0 Preparation of @met nanomaterials Weigh Fe 0 5 mg of nanomaterials and 50 mg of metformin were added to a glass bottle, followed by the addition of anhydrous ethanol. The mixture was ultrasonically dispersed and magnetically stirred at room temperature for 12 h. After the reaction was complete, the mixture was immediately centrifuged and the precipitate was collected. The precipitate was then vacuum dried at room temperature to obtain black Fe. 0 @met nanomaterials; Step 3: Fe 0 Preparation of @met-ss-SiO2 nanomaterials 3a. Add deionized water and ammonia to a glass bottle, mix well, and then add anhydrous ethanol to the glass bottle to obtain solution 1; solution 1 is composed of 1.35 mL of deionized water, 100 μL of ammonia and 3.75 mL of anhydrous ethanol. 3b. Weigh out 5mg of Fe 0 @met nanomaterials and 5mg amine-polyethylene glycol 2000 were added to a glass bottle, and then anhydrous ethanol was added to the glass bottle. The mixture was ultrasonically dispersed to obtain solution 2. 3c. Take tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide and add them to a glass bottle. Then add anhydrous ethanol to the glass bottle and disperse them evenly by ultrasonication to obtain solution 3. 3d. Add solutions 2 and 3 to solution 1 and sonicate for 2 h. After the reaction is complete, centrifuge and collect the precipitate. Wash with water and anhydrous ethanol respectively, and dry under vacuum at room temperature to obtain Fe. 0 @met-ss-SiO2 nanomaterials.

2. The method according to claim 1, characterized in that: In step 3c, the amount of tetraethyl orthosilicate added is 8 μL, and the amount of bis-[3-(triethoxysilyl)propyl]-disulfide added is 3.4 μL.

3. Fe prepared by any one of the preparation methods in claims 1-2 0 Application of @met-ss-SiO2 nanomaterials in the preparation of ROS inducers.

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