Active oxygen free radical generation method and application thereof

Abstract

The invention provides an active oxygen free radical generation method and application thereof. According to the method, a PFC nanoemulsion is utilized to catalyze H2O2 to generate active oxygen free radicals (hydroxyl radical.OH). The principle of generating reactive oxygen free radicals by the method is as follows: a PFC nanoemulsion and an H2O2 aqueous solution are adopted to form a liquid-liquid interface, interface contact is promoted through vortex or ultrasound, water molecules and hydroxyl ions are induced to lose electrons and generate. OH by utilizing a contact electrification effect, and PFC captures the electrons to form PFC * with negative electricity; then, H2O2 obtains electrons from PFC *, and OH is generated again. The method can be used in the fields of environmental pollutant degradation and drug preparation. The function and mechanism of the PFC nanoemulsion for catalyzing H2O2 to generate. OH active oxygen free radicals are found for the first time, the new application of the PFC nanoemulsion is widened, an important scientific basis is provided for developing a novel catalysis strategy, and the PFC nanoemulsion has important theoretical and application values.

Description

Technical Field

[0001] The invention relates to the field of environmental pollutant degradation and disease treatment, and in particular to a method for generating active oxygen free radicals and an application thereof. Background Art

[0002] Reactive oxygen species (ROS) are a class of highly reactive molecules, including hydroxyl radicals · OH, superoxide anion radicals · O2 - Excessive reactive oxygen radicals in cells can cause cell apoptosis through various mechanisms. Therefore, reactive oxygen radicals can be used to construct various disease treatment methods, such as photodynamic therapy (PDT), sonodynamic therapy (SDT), chemodynamic therapy (CDT), etc. However, when traditional reactive oxygen radical catalytic reactions are applied to biological systems, limited reaction substrates (O2, H2O2, etc.) and special biological environments (tumor microenvironment, etc.) limit the effective concentration of reactive oxygen radicals, severely inhibiting their biological activity. Exploring more efficient new mechanisms for the generation of reactive oxygen radicals and constructing new strategies for regulating reactive oxygen radical catalytic reactions are key challenges faced in breaking through the biological activity of reactive oxygen radicals.

[0003] Perfluorocarbon (PFC) is an inert chemical substance with extremely high fluorine content, high affinity for oxygen, and good biocompatibility. It has no reactivity in the human body and can be exhaled through the lungs or skin pores. These excellent properties make it widely used in clinical research and application as a potential artificial blood substitute. At present, most of the research on PFC is limited to using its excellent oxygen-carrying capacity to improve the tumor microenvironment. However, as an inert chemical substance, the catalytic activity of PFC itself has rarely been explored. Recently, our research found (J.Am.Chem.Soc.2024,146,12087-12099) that PFC nanoemulsion prepared with human serum albumin (HSA) as a stabilizer can catalyze water (including water molecules and hydroxide ions) to produce ·OH and ·O2 under ultrasonic conditions. - However, the catalytic efficiency of this method still needs to be improved, and whether PFC nanoemulsion has the potential to catalyze other substances has not yet been explored. Summary of the invention

[0004] The purpose of the present invention is to explore the catalytic potential of PFC nanoemulsion and provide a new method for generating active oxygen free radicals and its application. The method of the present invention utilizes a catalytic reaction system consisting of a PFC nanoemulsion modified with human serum albumin (HSA) and a hydrogen peroxide (H2O2) aqueous solution to generate ·OH under vortex or ultrasonic conditions. The mechanism of the catalytic reaction is as follows: Figure 1As shown in the figure: When the PFC nanoemulsion and the aqueous solution come into contact with the "liquid-liquid" interface, electron transfer occurs on the interface, that is, the fluorine atoms with stronger electronegativity are transferred from H2O or OH - The electrons are captured in the PFC, making it negatively charged (PFC*), while H2O or OH - Then, the electrons are lost and ·OH is generated; then, the H2O2 in the aqueous solution collides with the negatively charged PFC*, captures electrons from the PFC*, and generates ·OH again. The present invention provides a new method for generating active oxygen free radicals, and for the first time discovers the ability of PFC to catalyze H2O2 to produce ·OH. The present invention will broaden the new application of PFC nanoemulsion in the biomedical field, provide an important scientific basis for the development of new catalytic strategies, and has important theoretical and application value.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A novel method for generating active oxygen free radicals comprises the following steps: mixing a PFC nanoemulsion aqueous solution and a H2O2 aqueous solution to react and generate active oxygen free radicals. The active oxygen free radicals include ·OH.

[0007] Furthermore, the PFC is preferably perfluorotributylamine (PFTBA) with a high fluorine content.

[0008] Furthermore, the PFC nanoemulsion is a PFC nanoemulsion modified with human serum albumin (HSA), and HSA is used to stabilize the PFC nanoemulsion so that it can be stably stored in an aqueous solution. Figure 2 As shown, it is preferably prepared by a method comprising the following steps: adding PFC to an HSA aqueous solution and preparing a PFC nanoemulsion by ultrasound, wherein the concentration of the HSA aqueous solution is preferably 2-4 mg / mL, and the volume ratio of the PFC to the HSA aqueous solution is preferably 3:80-3:160.

[0009] Furthermore, in the catalytic reaction system composed of a mixture of an aqueous PFC nanoemulsion solution and an aqueous H2O2 solution, the concentration of the PFC nanoemulsion is preferably 0.1-0.5 mg / mL, and the concentration of the H2O2 is preferably 5-100 mM.

[0010] Further, the reaction of mixing the PFC nanoemulsion aqueous solution and the H2O2 aqueous solution is preferably carried out under vortex or ultrasound conditions, and the reaction temperature is preferably room temperature. The vortex or ultrasound promotes the "liquid-liquid" interface contact between the PFC nanoemulsion and the aqueous phase solution, causing electron transfer at the interface, and the electrons can be transferred from water and hydroxide ions to the PFC to produce ·OH, and the PFC carries a negative charge (PFC*). Subsequently, H2O2 obtains electrons from the negatively charged PFC (PFC*) to produce ·OH, realizing the process of PFC catalyzing H2O2 to produce ·OH.

[0011] The active oxygen free radical generation method is applied in the field of environmental pollutant degradation. The pollutants can react with active oxygen to be degraded, including methylene blue, terephthalic acid, etc.

[0012] A method for degrading environmental pollutants comprises the following steps: adding PFC nanoemulsion, H2O2 or a substance capable of producing H2O2 into water containing pollutants, and performing vortex or ultrasonic treatment.

[0013] The use of the reactive oxygen free radical generation method in the preparation of medicines, the medicines contain PFC nanoemulsions, and H2O2 or substances capable of producing H2O2, the substances capable of producing H2O2 include enzymes that produce H2O2 (e.g., glucose oxidase GOD), substances with enzyme-like (similar to enzymes that produce H2O2) activity (e.g., nanozymes), and medicines that promote the body to produce H2O2, etc. The indications of the medicines are diseases that can be treated by reactive oxygen, the diseases that can be treated by reactive oxygen include cancer, infectious diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular diseases, etc., the cancers include breast cancer, liver cancer, lung cancer, skin cancer, etc., the infectious diseases include bacterial inflammation, fungal infection, viral infection, etc., the autoimmune diseases include rheumatoid arthritis, multiple sclerosis, etc., the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, etc., the cardiovascular diseases include ischemic heart disease, peripheral arterial disease, etc.

[0014] Application of PFC nanoemulsion and H2O2 or substances capable of producing H2O2 in the preparation of environmental pollutant degradation agents or medicines.

[0015] An environmental pollutant degradation agent or drug comprises PFC nanoemulsion and H2O2 or a substance capable of generating H2O2.

[0016] Beneficial effects of the present invention:

[0017] (1) This paper provides a new method for generating reactive oxygen free radicals and discovers a new mechanism for PFC nanoemulsion to catalyze H2O2 to produce ·OH.

[0018] (2) The PFC used in the present invention is a clinical drug, which helps to promote the clinical application of this method.

[0019] (3) The preparation method of PFC nanoemulsion is simple and its catalytic performance is stable, which is conducive to clinical application.

[0020] (4) The PFC nanoemulsion prepared by the present invention can be applied to the field of environmental pollutant degradation and disease treatment.

[0021] (5) The present invention provides an important scientific basis for the development of new catalytic strategies and has important theoretical and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a mechanism diagram of the method for generating active oxygen radicals of the present invention.

[0023] Figure 2 It is a flow chart of the preparation of PFC nanoemulsion.

[0024] Figure 3 It is a transmission electron microscopy image of PFC nanoemulsion.

[0025] Figure 4 DMPO-·OH electron spin resonance spectra of PFC-H2O and PFC-H2O2 treated by vortex (a) or ultrasound (b).

[0026] Figure 5 It is the UV-visible absorption spectrum of methylene blue after vortex or ultrasonic treatment in the presence of PFC nanoemulsion-H2O2, and Control is the control group without any treatment.

[0027] Figure 6 It is the intracellular H2O2 level under GOD and PFC treatment.

[0028] Figure 7 Laser confocal scanning microscopy was used to detect the level of reactive oxygen species in 4T1 cells under different treatments. The reactive oxygen species probe was DCFH-DA. Scale bar: 50 μm.

[0029] Figure 8 is the cell survival rate of 4T1 cells under different treatments.

[0030] Fig. 9 is the cell survival rate of NALM6 cells under different treatments.

[0031] Fig.10 It is the volume ratio of the tumor in tumor-bearing mice in different treatment groups after 14 days of treatment to the tumor before treatment. DETAILED DESCRIPTION

[0032] The present invention is further described below by the following non-limiting examples. It should be understood that these examples are only some preferred technical solutions of the present invention, and the protection scope of the present invention is not limited to these examples.

[0033] Example 1 Preparation and Characterization of PFC Nanoemulsion

[0034] Preparation method: The preparation process of PFC nanoemulsion is as follows Figure 2 As shown, human serum albumin (HSA) and perfluorotributylamine (PFTBA) were prepared into PFC nanoemulsion by ultrasound, which specifically included the following steps: 16 mg of human serum albumin (HSA) was dissolved in 8 mL of H2O and stirred for 4 hours to form a 2 mg / mL HSA solution. Then, the obtained HSA solution was mixed with 300 μL of perfluorotributylamine (PFTBA) and stirred for 20 minutes, and then ultrasonicated for 20 minutes at 25°C using a cell crusher (SCIENTZ-ⅡD, China) to form a PFC nanoemulsion. The PFC nanoemulsion solution (5 mg / mL) was then collected by ultrafiltration centrifugation for use in the following examples.

[0035] Characterization: According to transmission electron microscopy, the PFC nanoemulsion prepared in this example has a typical spherical structure with an average size of about 95nm ( Figure 3 ).

[0036] Example 2 Evaluation of the performance of PFC nanoemulsion in catalyzing H2O2 to produce reactive oxygen free radicals

[0037] PFC nanoemulsion (0.2 mg / mL) was added to an aqueous solution containing H2O2 (100 mM) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 10 mM) or an aqueous solution containing DMPO (10 mM). The solution was then vortexed (700 rpm) or sonicated (30 kHz, 2.0 W / cm 2 ) for 10 minutes. The ESR spectrum of the mixed solution was measured by an electron paramagnetic resonance (ESR) spectrometer (Brukermagnettech ESR5000, Germany). According to the ESR test, PFC nanoemulsion can catalyze H2O2 to produce ·OH under vortex and ultrasonic conditions. Figure 4 ). Moreover, the amount of ·OH produced by PFC catalyzing H2O2 is significantly increased by 2.3 times compared with the amount of ·OH produced by PFC alone.

[0038] Example 3 PFC nanoemulsion is used for the degradation of environmental pollutant methylene blue (MB)

[0039] PFC nanoemulsion was added to an aqueous solution containing H2O2 and MB, and the UV-visible absorption spectrum of MB was recorded after ultrasonic or vortex treatment. The specific experimental process is as follows: PFC nanoemulsion (0.2 mg / mL) was added to an aqueous solution containing H2O2 (100 mM) and MB (10 μg / mL) with a total volume of 3 mL. Vortex (700 rpm) or ultrasound (30 kHz, 2.0 W / cm 2 ) for 10 minutes. Subsequently, the mixed solution was subjected to ultrafiltration centrifugation (10000 rpm). Finally, the absorbance change of MB was measured by UV-visible absorption spectroscopy. The results are shown in Figure 5 Under the treatment of vortexing for 10 minutes, MB was degraded due to the degradation of H2O2 and the generation of ·OH, resulting in a decrease in the absorbance at 664nm, with a degradation rate of 41.82%. Under the same conditions of ultrasonic treatment, the decrease in the absorbance of MB was more significant, with a degradation rate of 74.64%. The ability of PFC nanoemulsion to catalyze H2O2 to produce reactive oxygen species was demonstrated, and ultrasound induced a higher frequency of electron exchange in the PFC-water "liquid-liquid" interface, generating more reactive oxygen free radicals.

[0040] Example 4: Increasing the level of H2O2 and combining it with PFC nanoemulsion to increase the content of intracellular reactive oxygen free radicals

[0041] Glucose oxidase (GOD) was used to increase the intracellular H2O2 content. First, 4T1 cells were cultured at 8×10 4 The cells were inoculated into 6-well plates at a density of 15 μg / mL and incubated for 12 h. Then, PFC nanoemulsion (15 μg / mL) or glucose oxidase (0.2 μg / mL) was added and incubated for 24 h. The concentration of H2O2 in the cells was detected using a H2O2 detection kit. The results are shown in Figure 6 Since glucose oxidase can catalyze glucose in cells to produce H2O2, the H2O2 content in 4T1 cells increased significantly by 2.2 times after incubation with glucose oxidase.

[0042] The probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to monitor the level of reactive oxygen free radicals produced by cells stimulated by PFC nanoemulsion. 4T1 cells were cultured at 8×10 4The cells were inoculated into 6-well plates at a density of 10 cells / well and cultured for 12 hours in a total volume of 2 mL. The experimental groups were Control, GOD, PFC, and PFC+GOD. Among them, the Control group did not receive any treatment, the GOD group added glucose oxidase (0.2 μg / mL) to the culture medium and incubated for 24 hours, the PFC group added PFC nanoemulsion (15 μg / mL) to the culture medium and incubated for 24 hours, and the PFC+GOD group added glucose oxidase (0.2 μg / mL) and PFC nanoemulsion (15 μg / mL) to the culture medium and incubated for 24 hours. For the ultrasonically treated group, the same grouping and treatment scheme were set up as above, and then ultrasonic waves (30 kHz, 2.0 W / cm 2 , 10 minutes) to treat the cells. After 24 hours of co-culture, DCFH-DA (10 μM) was added to stain the reactive oxygen species of the cells for 15 minutes. The fluorescence generated by 4T1 cells after DCFH-DA staining was measured using a confocal laser scanning microscope. After entering the cells, the non-fluorescent reactive oxygen probe DCFH-DA probe can be hydrolyzed by intracellular esterase to produce DCFH, and then the reactive oxygen in the cells can oxidize DCFH to produce green fluorescent DCF, thereby realizing the detection of intracellular reactive oxygen species. The results are shown in Figure 7 As shown, under ultrasound treatment, tumor cells co-incubated with PFC nanoemulsion and glucose oxidase produced stronger green fluorescence than cells incubated with PFC nanoemulsion alone, proving that when glucose oxidase increased the content of intracellular H2O2, PFC nanoemulsion could catalyze H2O2 in 4T1 cells under ultrasound to cause the production of more reactive oxygen free radicals.

[0043] Example 5: Increasing H2O2 levels and combining PFC nanoemulsion to kill tumor cells

[0044] 4T1 cells and NALM6 cells were cultured at a rate of 5×10 3 The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured for 12 h. PFC nanoemulsion (15 μg / mL), GOD (0.2 μg / mL), and GOD (0.2 μg / mL) + PFC nanoemulsion (15 μg / mL) were added to the culture medium, and then ultrasonic waves (30 kHz, 2.0 W / cm 2 After 24 hours of co-culture, the relative cell viability of 4T1 cells and NALM6 cells was measured by MTT method. Figure 8 , 9As shown in the figure, under ultrasonic treatment, the survival rates of 4T1 cells and NALM6 cells in the control group were 97.2% and 99.5%, respectively, while the survival rates of tumor cells in the experimental group co-cultured with PFC nanoemulsion were greatly reduced, 74.8% and 45.5%, respectively. The reason is that ultrasound-induced PFC nanoemulsion catalyzes H2O2 in the cells to produce reactive oxygen free radicals (·OH), causing the death of 4T1 cells and NALM6 cells. In addition, after adding GOD, since GOD increases the content of H2O2 in the cells, PFC nanoemulsion can catalyze more H2O2 to produce a large number of reactive oxygen free radicals under ultrasound, further promoting the death of 4T1 cells, and the cell survival rates are as low as 23.7% and 31.6%, respectively.

[0045] Example 6: Upregulation of H2O2 levels combined with PFC nanoemulsion for breast cancer treatment in 4T1 tumor-bearing mice

[0046] The BALB / c mouse breast cancer model was established using the 4T1 breast cancer cell line. When the mouse tumor grew to 100 mm 3 Around V0, PFC nanoemulsion was used for treatment. The administration method was intratumoral injection. The injection interval was once every 2 days. The single injection dose of glucose oxidase (GOD) was 50 μg / kg, the single injection dose of PFC nanoemulsion was 2 mg / kg, and the time of each ultrasound treatment (US) was 10 minutes (30 kHz, 2.0 W / cm 2 The experimental groups included: control group (Control), ultrasound treatment control group (US) (PBS injection, dose: 100 μL), GOD group, GOD+US group, PFC group, PFC+US group and PFC+GOD+US group. The tumor volume (V) of mice was recorded after 14 days of treatment. Fig.10 As shown, the tumors of mice in the Control group, US group, GOD group, PFC group, PFC+GOD group, and GOD+US group were larger after 14 days of treatment. However, after combined ultrasound treatment, the volume ratio (V / V0) of the tumors of mice in the PFC+US and PFC+GOD+US groups to the tumors before treatment was 4.9 and 0.28, respectively, which was significantly less than 12.9 in the Control group, proving that tumor growth was significantly inhibited. In particular, the tumor inhibition effect of the PFC+GOD+US group was significantly better than that of the PFC+US group, proving that the addition of GOD oxidase significantly enhanced the tumor treatment effect. This is because GOD increases the H2O2 content in the tumor site, allowing PFC nanoemulsion to catalyze H2O2 to produce more reactive oxygen free radicals under ultrasound, thereby enhancing the tumor treatment effect.

[0047] The above description is only a preferred specific implementation manner of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the scope of protection of the invention.

Claims

1. A method for generating active oxygen free radicals, characterized in that: The following steps are involved: The PFC nanoemulsion aqueous solution and the H2O2 aqueous solution are mixed and reacted to generate active oxygen free radicals, wherein the active oxygen free radicals include ·OH.

2. The method for generating active oxygen free radicals according to claim 1, characterized in that: The PFC is perfluorotributylamine.

3. The method for generating active oxygen free radicals according to claim 1, characterized in that: The PFC nanoemulsion is a PFC nanoemulsion modified with human serum albumin.

4. The method for generating active oxygen free radicals according to claim 1, characterized in that: The PFC nanoemulsion aqueous solution and the H2O2 aqueous solution are mixed and reacted under vortex or ultrasonic conditions.

5. Application of the method for generating active oxygen free radicals according to any one of claims 1 to 4 in the field of environmental pollutant degradation, characterized in that: The pollutants can react with active oxygen to be degraded.

6. A method for degrading environmental pollutants, characterized in that: The following steps are involved: PFC nanoemulsion, H2O2 or a substance capable of generating H2O2 is added to water containing pollutants, and vortex or ultrasonic treatment is performed.

7. Use of the method for generating active oxygen free radicals according to any one of claims 1 to 4 in the preparation of medicines, characterized in that: The medicine comprises PFC nanoemulsion and H2O2 or a substance capable of producing H2O2; the medicine is indicated for diseases that can be treated by active oxygen.

8. The use according to claim 7, characterized in that: The diseases that can be treated by active oxygen include cancer, infectious diseases, autoimmune diseases, neurodegenerative diseases, and cardiovascular diseases.

9. The use of PFC nanoemulsion and H2O2 or a substance capable of producing H2O2 in the preparation of a medicine, characterized in that: The drug is indicated for diseases that can be treated by active oxygen.

10. An environmental pollutant degradation agent or drug, characterized in that: Contains PFC nanoemulsion, and H2O2 or a substance capable of generating H2O2.

Images