CN113042076B - Catalase activity-simulated photocatalytic nanoenzyme, and preparation method and application thereof - Google Patents
Catalase activity-simulated photocatalytic nanoenzyme, and preparation method and application thereof Download PDFInfo
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Images
Classifications
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- B01J35/39—
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/26—Phosphorus; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J27/00—Catalysts comprising the elements or compounds of halogens, sulfur, selenium, tellurium, phosphorus or nitrogen; Catalysts comprising carbon compounds
- B01J27/14—Phosphorus; Compounds thereof
- B01J27/186—Phosphorus; Compounds thereof with arsenic, antimony, bismuth, vanadium, niobium, tantalum, polonium, chromium, molybdenum, tungsten, manganese, technetium or rhenium
- B01J27/188—Phosphorus; Compounds thereof with arsenic, antimony, bismuth, vanadium, niobium, tantalum, polonium, chromium, molybdenum, tungsten, manganese, technetium or rhenium with chromium, molybdenum, tungsten or polonium
- B01J27/19—Molybdenum
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B25/00—Phosphorus; Compounds thereof
- C01B25/08—Other phosphides
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/03—Particle morphology depicted by an image obtained by SEM
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/62—Submicrometer sized, i.e. from 0.1-1 micrometer
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/30—Wastewater or sewage treatment systems using renewable energies
- Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy
Abstract
The invention relates to the field of medical treatment and health, in particular to a photocatalytic nanoenzyme with catalase activity imitation, a preparation method and application thereof. The photocatalytic nanoenzyme consists of MoP2Crystals prepared, the MoP prepared2The nano particles are irradiated by near infrared laser to obtain the nano particles with H2O2Photocatalytic nanoenzyme with catalytic activity for catalyzing H2O2Rapidly generate a large amount of active free radicals such as hydroxyl free radicals (. OH). Infrared laser-mediated MoP compared to existing catalase2The nanoparticles can efficiently catalyze H in a complex microenvironment without the use of catalase2O2The preparation has wide application in the medical and health fields of tumor treatment, antibacterial agent, sterilization, disinfection and the like.
Description
Technical Field
The invention belongs to the technical field of medical treatment and health, and particularly relates to a catalase activity-simulated photocatalytic nanoenzyme, and a preparation method and application thereof.
Background
Catalase (also called Catalase, CAT) is a kind of terminal oxidase widely existing in animals, plants and microorganisms, and Hydrogen peroxide is used as a substrate to catalyze the transfer of a pair of electrons to finally decompose the Hydrogen peroxide into water and oxygen. Catalase in food, medicine and textileThe method has important application in industries such as papermaking and environmental protection. When catalase is applied to the treatment of organic tissue, its activity is influenced by temperature, pH, H2O2The influence of micro environments such as content and the like limits the application of the nano-particles in the field of medical health.
The nano enzyme is a mimic enzyme which not only has the unique performance of nano materials, but also has a catalytic function. The nano enzyme has the characteristics of high catalytic activity of natural enzyme and stability and economy of simulated enzyme, so that the research of the nano enzyme is rapidly increased since the report of the HRP nano enzyme in 2007, the related aspects of the research are gradually wide, and the research comprises different fields of material science, physics, chemistry, biology, medicine, environment and the like. The emergence of nanoenzymes provides a new method for the diagnosis of tumors. Utilizes the specificity recognition and high catalytic activity of tumor cells of nano-enzyme to catalyze over-expressed H in tumor focus area2O2Generating strongly oxidative active species such as hydroxyl radical (. OH) to induce apoptosis of tumor cells. Since OH with a certain concentration can destroy biological macromolecules of bacteria, including DNA, cell protein, membrane lipid and the like, the sterilization effect is achieved. Therefore, the method has wider application prospect in tumor treatment. However, the catalytic efficiency of nanoenzymes in complex tumor microenvironments is limited, so that the amount of OH radicals generated is very small and affects the therapeutic effect. The production amount required for the OH free radical treatment of tumor needs to be increased, which can cause some uninhibitable side effects, such as nephrotoxicity and severe anaphylaxis. Therefore, there is an urgent need to find nanoenzymes having high catalytic activity in a complex microbial environment.
MoP2The material has the unique properties of small effective mass, high carrier mobility, small energy band overlapping energy and the like. At present, the research is mainly used in the field of photoelectrocatalysis, and MoP is not seen yet2The application of the nano enzyme in medical health is reported. MoP2The nano-particles have good biocompatibility, photo-thermal conversion efficiency and peroxide-like activity, and have extremely strong H under near infrared irradiation2O2Catalytic activity, capable of catalyzing H2O2Rapidly generate strong oxidizing active substances such as hydroxyl free radical (. OH)The method can induce tumor cell apoptosis and kill bacteria in tumor microorganism environment. In a complex in vivo environment, the MoP2The nanoparticles can be gradually degraded into free metal ions and non-toxic phosphates. Since phosphorus is an essential element for human body, it is present in large amount (1%) in human body and molybdenum is a trace element, MoP2The degradation product of the nano-particles can be absorbed or discharged by human body, and has better biological safety. Thus, the photocatalytic MoP used in the present invention2The nano-particles can be used as catalase-imitating nano-enzyme in the field of medical treatment and health.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method and application of a photocatalytic nanoenzyme with catalase activity. The preparation method is simple, and the prepared photocatalytic nanoenzyme has high H2O2Catalytic activity, excellent stability and good biocompatibility. Can catalyze H rapidly under illumination2O2The generation of hydroxyl free radicals (. OH) can cause the apoptosis of tumor cells by destroying biological molecular substances such as lipids, nucleic acids, proteins and the like of the cells. Meanwhile, the high concentration OH can quickly decompose substances such as zymotin, RNA, lysozyme and the like contained in the bacteria, thereby achieving better antibacterial effect. The photocatalytic nanoenzyme has great application potential in the medical and health fields of tumor treatment, sterilization, disinfection and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method and application of a photocatalytic nanoenzyme with catalase activity. The photocatalytic nanoenzyme consists of MoP2And (4) preparing crystals. The MoP2The crystal is formed by red phosphorus and molybdenum through the interaction of high-temperature reaction and rapid cooling in a vacuum sealing environment. The mass ratio of the red phosphorus to the molybdenum is 1.69-4: 1.
Further, the high-temperature reaction temperature is between 600 ℃ and 1000 ℃ and lasts for 16-18 hours.
Further, the cooling speed is 1-3 ℃/min, and the temperature is cooled to 20-35 ℃.
The invention also provides a preparation method of the photocatalytic nanoenzyme, which comprises the following steps:
(1) mixing the above MoP2Centrifuging after carrying out ultrasonic treatment on the crystals in a solvent, and collecting supernatant;
(2) centrifuging the supernatant twice, taking the precipitate, and washing for several times to obtain MoP2Nanoparticles, re-suspended in the dispersion;
(3) irradiation of the MoP with near-infrared laser2And (4) nano particles to obtain the photocatalytic nano enzyme. Fast catalysis of H2O2Active oxides such as hydroxyl radical (. OH) are generated.
Further, in the preparation method, the solvent in the step (1) is one of N-methyl-2-pyrrolidone, N-dimethylformamide, N-butanol or ethanol;
further, in the preparation method, the ultrasonic treatment time in the step (1) is 5-10 h;
further, in the preparation method, the centrifugal speed in the step (1) is 2000-4000 rpm, and the centrifugal time is 10-20 min;
further, in the preparation method, the secondary centrifugation speed in the step (2) is 10000rpm, and the centrifugation time is 10-20 min;
further, in the preparation method, the MoP in the step (2)2The nano particles are irregular, the transverse diameter is 100-500 nm, and the longitudinal diameter is 50-200 nm.
Further, in the preparation method, the dispersion liquid in the step (2) can be water, ethanol, PBS buffer solution, 0.9% physiological saline for injection;
further, in the preparation method, the dispersion liquid in the step (2) disperses the MoP2The concentration of the nano particles is 20-50 mu g mL-1。
Further, in the preparation method, the wavelength of the near-infrared laser in the step (3) is 780-2526 nm.
Further, in the preparation method, the MoP is irradiated after the near-infrared laser in the step (3)2The temperature of the nano-particle dispersion liquid is less than or equal to45℃。
Further, the photocatalytic nanoenzyme has extremely high H2O2High catalytic efficiency and can quickly catalyze H2O2Active oxides such as hydroxyl radical (. OH) are generated.
The invention also provides application of the photocatalytic nanoenzyme in medical treatment and health.
Further, the application is applied to tumor treatment.
The photocatalytic nanoenzyme is administered in different doses depending on the clinical condition of the cancer patient diagnosed early. The photocatalytic nanoenzyme has extremely strong stability in tumor focus areas with complex microbial environments. Real-time excitation of H by near-infrared illumination2O2Catalytic activity, rapid catalysis of H in the focal zone of tumors2O2Active oxides such as hydroxyl free radical (. OH) are generated, and the tumor cells are effectively killed.
Further, the application is applied to sterilization and disinfection.
The photocatalytic nanoenzyme has high catalytic activity under near infrared light irradiation and can rapidly catalyze H2O2The generated active oxides such as hydroxyl free radical (. OH) and the like can effectively and quickly sterilize and disinfect. The dosage is small, and the sterilization efficiency is high.
The invention has the following technical characteristics:
(1) the photocatalytic nanoenzyme has high H under the irradiation of infrared light2O2The catalyst has high catalytic efficiency, solution stability, photodynamic stability and high active oxygen production rate, and has wide application.
(2) The photocatalytic nanoenzyme has better biocompatibility. The molybdenum can be gradually degraded into free molybdenum ions and nontoxic phosphate in a human body, the molybdenum element is a trace element necessary for the human body, and the molybdenum ions and the phosphate are harmless to the human body.
(3) The photocatalytic nanoenzyme provided by the invention has the advantages of simple preparation method, high catalytic efficiency and small dosage, and is simple and effective in application in the medical and health fields of tumor treatment, sterilization, disinfection and the like.
Drawings
FIG. 1 shows MoP prepared in example 1 of the present invention2Transmission electron microscopy of nanoparticles;
FIG. 2 shows the MoP prepared in example 1 of the present invention2Catalysis of H by nano particles under induction of infrared laser2O2A resolved activity map;
FIG. 3 shows MoP prepared in example 1 of the present invention2The efficiency graph of the nanoparticles catalyzing the generation of hydroxyl radicals (. OH) under the induction of infrared laser;
FIG. 4 shows MoP prepared in example 1 of the present invention2A cytotoxicity test profile of the nanoparticles;
FIG. 5 shows MoP prepared in example 1 of the present invention2Comparing the performance of the anti-tumor cells of the nanoparticles under the induction of infrared laser;
FIG. 6 shows MoP prepared in example 1 of the present invention2A test chart of the survival rate of the escherichia coli with or without the induction of the infrared laser irradiation on the nanoparticles;
FIG. 7 shows MoP prepared in example 1 of the present invention2A staphylococcus aureus survival rate test chart of the nanoparticles under the induction of infrared laser irradiation or not;
FIG. 8 shows MoP prepared according to example 1 of the present invention2SEM appearance change graphs of the Escherichia coli and the staphylococcus aureus of the nanoparticles under the induction of infrared laser irradiation.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.
Example 1:
100mg of molybdenum powder and 300mg of red phosphorus blocks are weighed by a balance, placed at one end of a quartz tube and sealed in vacuum. The quartz tube was horizontally placed in a muffle furnace, heated at 850 ℃ for 18 hours for high-temperature reaction, and then cooled to 30 ℃ at a rate of 2 ℃/min. MoP formed by taking out hot end of quartz tube2The crystal is treated by ultrasonic treatment in N-methyl-2-pyrrolidone (NMP) for 10 hours,centrifuging at 4000rpm for 10min to obtain MoP2And (3) nanoparticles. Collecting the mixture containing MoP2The supernatant of the nanoparticles was centrifuged at 10000rpm for 15 minutes. The resulting pellet was washed twice with ethanol and resuspended in PBS buffer for further use.
Example 2:
MoP prepared in example 12The nano particles are irregular, the transverse diameter is 100-400 nm, and the length is 80-100 nm. Has good H under the irradiation of infrared laser2O2High catalytic activity and stability, and high catalytic activity of H2O2Forming active oxides such as hydroxyl free radical (. OH) and the like, and is applied to the medical and health fields such as tumor treatment, sterilization, disinfection and the like.
The MoP prepared in example 1 was observed using a Transmission Electron Microscope (TEM)2Morphology and nanometer size of nanoparticles, and application of the nanoparticles to infrared irradiation2O2The catalytic activity was characterized and the formation of hydroxyl radicals (. OH) in solution was monitored. The specific test results are as follows:
(1) transmission Electron Microscope (TEM)
Transmission Electron Microscope (TEM) representation of the MoP prepared2Morphology and nanometer size of the nanoparticles. Results referring to FIG. 1, detection of MoP at 200nm is shown2The distribution and morphology of the nanoparticles, from FIG. 1, the MoP can be illustrated2The nanoparticles exhibit an irregular rod-like structure.
(2)H2O2Catalytic efficiency
CAL27 xenogeneic oral tumor cells and SCC-9 human tongue squamous carcinoma cells were seeded in 96-well plates (approximately 1104 cells per well) and incubated overnight. Setting a cell experiment group: a is a control group (H)2O2) B is MoP2Nanoparticle catalytic group (H)2O2+MoP2) C is infrared light catalytic group (H)2O2+ NIR), d is infrared light mediated MoP2Nanoparticle catalytic group (H)2O2+MoP2+NIR)。
The specific implementation operation is as follows: each group was added with 100. mu. mol mL-1H of (A) to (B)2 O 240. mu.g mL of the additive was added to the groups b and d-1MoP2Incubating nanoparticles, and irradiating group c and group d with near infrared radiation (0.5W cm)-2) After 10 minutes, a hydrogen peroxide detection reagent (Beyotime Biotech, Shanghai, China) was added to each set of samples according to the instructions, 50. mu.L of the sample was mixed with 100. mu.L of the reagent at room temperature for 30 minutes, and then the absorbance was measured at 560nm with a microplate reader (Multisken sky, ThermoFisher, China). Calculation of H in each group using the standard curve of the standard solution2O2Concentration (. mu. mol mL)-1)。
Results referring to FIG. 2, the results show that the near infrared light irradiation of MoP is compared with the control group2Condition of nanoparticles H2O2The content of (a) is drastically reduced. While using MoP alone2Nanoparticles or irradiation of H with near-infrared light alone2O2The content does not have too great an effect. Representing near infrared light activated MoP2The nano particles have strong catalytic activity and can effectively catalyze H2O2Decomposition of (3).
(3) Efficiency of production of hydroxyl radical (. OH)
CAL27 xenogeneic oral tumor cells and SCC-9 human tongue squamous carcinoma cells were seeded in 96-well plates (approximately 1104 cells per well) and incubated overnight. Setting a cell experiment group: a is H2O2B is MoP2Nanoparticles, c is MoP2Nanoparticles and H2O2(MoP2+H2O2) D is near infrared photocatalysis MoP2Nanoparticles (MoP)2+ NIR) and e are near infrared photocatalysis H2O2(H2O2+ NIR), f is H2O2Infrared photocatalytic MoP in environment2Nanoparticles (MoP)2+H2O2+NIR)。
The specific implementation operation is as follows: add 100. mu. mol mL to groups a, c, e, f-1ofH2O240. mu.g mL of the solution was added to the groups b, c, d and f-1MoP2Incubating nanoparticles, and irradiating groups d, e and f with near infrared radiation (0.5W cm)-2) After 10 minutes, each set of media and trypsinized cells was collected, and pooled with 500. mu. mol mL-1Terephthalic Acid (TA) was mixed, and the solution was placed in an orbital incubator at 37 ℃ and gently shaken in the dark for 12 hours, and then the change in the fluorescence emission peak at 435nm was measured by fluorescence spectroscopy (F-4600, Hitachi, Japan). The percent enhancement was calculated as (Ftest Fblank)/(Fcontrol Fblank), where Fblank is the initial amount of fluorescence intensity in the presence of TA.
Results refer to FIG. 3, which shows MoP2Nanoparticles in H2O2MoP activated by near infrared light and capable of generating a small amount of hydroxyl free radicals in environment2Nanoparticles in H2O2The large amount of hydroxyl free radicals can be generated in the environment, which indicates that the MoP is photocatalytic2The nano particles have extremely strong catalase activity and can quickly and efficiently catalyze H2O2Decomposition produces a large amount of hydroxyl radicals (. OH).
Example 3:
this example is for the MoP prepared according to the invention2The cytotoxicity of the nanoparticles was evaluated.
Culturing heterogeneous oral tumor cell (CAL27), human tongue squamous carcinoma cell (SCC9) and Human Oral Keratinocyte (HOK) in DMEM medium containing 10% fetal calf serum at 37 deg.C and 5% CO2The culture is carried out in a humidified atmosphere. MoP was evaluated by the standard cell counting kit-8 (CCK-8) method2Cytotoxicity of the nanoparticles.
The specific implementation operation is as follows: the xenogenic oral tumor cells (CAL27), human tongue squamous carcinoma cells (SCC9) and Human Oral Keratinocyte (HOK) were placed in 48-well plates (2X 10) with 400. mu.L of complete medium, respectively4Cell/well), 5% CO at 37 ℃ respectively2And then the mixture is incubated for 24 h. The medium was then replaced with 200. mu.L of fresh complete medium containing different concentrations of MoP2Nanoparticles, after 24 hours of incubation, were removed from the medium, washed three times with Phosphate Buffered Saline (PBS), added 10. mu.L of the cell counting kit CCK-8 reagent and further incubated at 37 ℃ for 1.5 h. Subsequently, the Optical Density (OD) of the living cells at 450nm was determined with a microplate reader (Varioskan Flash 4.00.53, Finland). Cell survival (%) - (ODtest-ODblank)/(ODcontrol-ODblank) 100%.
Results referring to FIG. 4, the MoP is shown at different concentrations2The added nanoparticles have good cell compatibility and high cell survival rate on heterogeneous oral tumor cells (CAL27), human tongue squamous carcinoma cells (SCC9) and Human Oral Keratinocyte (HOK).
Example 4:
this example is for the MoP prepared according to the invention2The effect of the nanoparticles on tumor cells under near-infrared light irradiation is evaluated.
Culturing heterogeneous oral tumor cell (CAL27) and human tongue squamous carcinoma cell (SCC9) in DMEM medium containing 10% fetal calf serum at 37 deg.C and 5% CO2The culture is carried out in a humidified atmosphere. The biocompatibility of the nanosphere is detected by a live/dead cell staining kit (Calcein AM/PI), the characteristic that Calcein AM is an excellent fluorescent staining agent for live cells and can easily penetrate through the live cells is utilized, and when the Calcein AM reaches the cells, the Calcein AM can be hydrolyzed by esterase to be Calcein which is remained in the cells and shows strong green fluorescence; propidium Iodide (PI) in the kit cannot cross live cell membranes, but can cross disordered regions of dead cell membranes to reach cell nuclei, and generates red fluorescence (excitation wavelength of 535nm and emission wavelength of 617nm) after being embedded into DNA helices of cells so that the dead cells show red fluorescence.
Setting a cell experiment group: a is a blank control group; b is MoP2Nanoparticle dispersion (40. mu.g mL)-1) (ii) a c is H2O2Solution (100. mu.M), d is MoP2Nanoparticle dispersion (40. mu.g mL)-1) And H2O2Solution (100. mu.M). All experimental groups used a near infrared laser (1W/cm) with a wavelength of 808nm2) And irradiating for 10 minutes or not, and performing cell alive-death characterization after the treatment is finished.
The specific implementation operation is as follows: respectively taking heterogenous oral tumor cell (CAL27) and human tongue squamous carcinoma cell (SCC9) at logarithmic growth phase at 5 × 104and/mL, adding 400 mu L of each well into a 48-well plate, culturing in a cell culture box for 24h, then gently sucking out the cell culture medium in each well, adding 400 mu L of cell culture medium containing different components to be detected into each well, and continuing culturing for 24h, wherein the serum-free medium is used as a blank control. Taking out 48-hole cells for cultureThe plates, the well medium was aspirated off, the well plates were carefully washed 3 times with PBS solution, then calcein and propidium iodide solution was added to each well, after incubation for 20 minutes in the incubator, the dye was aspirated off and carefully washed 3 times with PBS solution and placed under a fluorescence microscope for observation and counting.
Results referring to FIG. 5, showing MoP2The nano-particles can effectively induce the apoptosis of heterogeneous oral tumor cells (CAL27) and human tongue squamous carcinoma cells (SCC9) under near-infrared illumination. But has good biocompatibility under the condition of no near infrared illumination.
Example 5:
this example is for the MoP prepared according to the invention2The antibacterial effect of the nanoparticles under near-infrared irradiation was evaluated.
Escherichia coli and staphylococcus aureus are used as bacterial models, and experimental groups are set as follows: a is a blank control group; b is MoP2Nanoparticle dispersion (40. mu.g mL)-1) (ii) a c is H2O2Solution (100. mu.M), d is MoP2Nanoparticle dispersion (40. mu.g mL)-1) And H2O2Solution (100. mu.M). All experimental groups used a near infrared laser (1W/cm) with a wavelength of 808nm2) Irradiation for 10 minutes or none.
(1) Plate counting method
The specific implementation operation is as follows: selecting a small amount of bacteria conserving solution, adding into a bacteria culture medium containing LB liquid, placing in a bacteria shaking tube at 37 deg.C, shaking in a 240rmp bacteria constant temperature incubator overnight for culture, collecting bacteria with a refrigerated centrifuge (5000rmp, 2min), washing with sterile normal saline, adjusting bacteria concentration with normal saline, detecting with a multifunctional microplate reader until the bacteria suspension concentration reaches OD600 of 0.01 (0.4-0.5) x 106cfu/mL)。
And mixing 100 mu L of each sample with 100 mu L of activated bacterial suspension, uniformly coating the mixture on the surface of a culture dish, uniformly coating the bacterial liquid on the culture dish, placing the culture dish in a bacterial incubator, culturing for 18 hours at 37 ℃ under constant temperature and humidity, and observing the number of bacterial colonies. The light set used 808nm (1W/cm)2) After the near infrared laser is irradiated for 10min, the bacteria liquid is uniformly coated on the culture mediumAfter culturing the culture dish, placing the culture dish in a bacterial incubator for culturing for 18h at the constant temperature and humidity of 37 ℃, and observing the number of colonies. Each sample was replicated 5 times in parallel. Blank control group sample solution was changed to equal volume of saline.
Results refer to FIGS. 6 and 7, which show H2O2MoP in the Environment2The nano-particles can effectively kill escherichia coli and staphylococcus aureus under near-infrared illumination, and have no great killing effect on bacteria under the condition of no near-infrared illumination.
(2) Bacterial SEM morphology
The morphological changes of the escherichia coli and the staphylococcus aureus after different treatments were observed by a Scanning Electron Microscope (SEM). The specific real-time operation is as follows: the bacterial pellet was dispersed in 2.5% glutaraldehyde solution and fixed overnight at 4 ℃. The fixed bacteria were washed 3 times with PBS, dehydrated sequentially with 25, 50, 80, 100 wt% ethanol for 10min, and completely dried at room temperature. Then, the bacteria were sputtered with gold (30s,30mA) and observed by Zeiss Sigma 300 scanning electron microscope.
Results refer to FIG. 8, representation H2O2MoP in the Environment2The nano-particles can distort the shapes of escherichia coli and staphylococcus aureus and obviously shrink the membrane structure under the near-infrared illumination, and the two bacteria keep the original shapes under the condition of no near-infrared illumination.
The applicant states that the invention is illustrated by the above examples to show the preparation of catalase-imitating photocatalytic nanoenzyme and its application scheme. The invention is not limited to the above examples, and equivalent substitutions of raw materials, addition of auxiliary components, selection of specific modes and the like of the product of the invention are all within the protection scope and the disclosure of the invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (18)
1. A catalase-imitated photocatalytic nanoenzyme is characterized in that: the photocatalytic nanoenzyme consists of MoP2Obtained by the preparation of crystals, MoP2Preparation of crystals to obtain MoP2Nanoparticles, MoP prepared2The nano particles are irradiated by near infrared laser to obtain the nano particles with H2O2A catalytically active photocatalytic nanoenzyme.
2. The catalase-like photocatalytic nanoenzyme of claim 1, wherein: the MoP2The crystal is formed by red phosphorus and molybdenum through the interaction of high-temperature reaction and rapid cooling in a vacuum sealing environment.
3. The catalase-like photocatalytic nanoenzyme of claim 2, wherein: the mass ratio of the red phosphorus to the molybdenum is (1.69-4) to 1.
4. The catalase-like photocatalytic nanoenzyme of claim 2, wherein: the high-temperature reaction temperature is 600-1000 ℃, and the reaction time is 16-18 h.
5. The catalase-like photocatalytic nanoenzyme of claim 2, wherein: the cooling speed is 1-3 ℃/min, and the temperature is cooled to 20-35 ℃.
6. The catalase-imitated photocatalytic nanoenzyme according to any one of claims 1 to 5, wherein: the photocatalytic nanoenzyme is in an irregular shape.
7. The method for preparing catalase-imitated photocatalytic nanoenzyme according to any one of claims 1 to 6, wherein the method comprises the following steps: the method comprises the following steps:
(1) adding MoP2Centrifuging after carrying out ultrasonic treatment on the crystals in a solvent, and collecting supernatant;
(2) centrifuging the supernatant twice, taking the precipitate, and washing for several times to obtain MoP2Nanoparticles, re-suspended in the dispersion;
(3) irradiation of the MoP with near-infrared laser2Nanoparticles of obtaining a compound having H2O2A catalytically active photocatalytic nanoenzyme.
8. The method according to claim 7, wherein in the step (1), the solvent is one of N-methyl-2-pyrrolidone, N-dimethylformamide, N-butanol or ethanol; the ultrasonic treatment time is 5-10 h, the centrifugal speed is 2000-4000 rpm, and the centrifugal time is 10-20 min.
9. The preparation method according to claim 7, wherein in the step (2), the secondary centrifugation speed is 10000rpm, and the centrifugation time is 10-20 min; the dispersion is selected from one of water, ethanol, PBS buffer solution and 0.9% physiological saline for injection.
10. The method according to claim 7, wherein the dispersion comprises MoP2The concentration of the nano particles is 20-50 mu g mL-1。
11. The preparation method according to claim 7, wherein in the step (3), the wavelength of the near-infrared laser is 780-2526 nm.
12. The method according to claim 11, wherein the wavelength of the near-infrared laser is 780 to 1100 nm.
13. The preparation method according to claim 7, wherein in the step (3), the irradiation time of the near-infrared laser is 10 to 30 min.
14. The preparation method according to claim 7, wherein the temperature of the photocatalytic nanoenzyme dispersion after the infrared laser irradiation in the step (3) is not more than 45 ℃.
15. An antibacterial material comprises a photocatalytic nanoenzyme (MOP)2Obtained by the preparation of crystals, MoP2Preparation of crystals to obtain MoP2Nanoparticles, MoP prepared2The nano-particles are irradiated by near-infrared laser to obtain the photocatalytic nano-enzyme with antibacterial property.
16. Use of the photocatalytic nanoenzyme according to any one of claims 1 to 6 for the preparation of a medicament for treating tumors.
17. The use of claim 16, the use of the photocatalytic nanoenzyme in the preparation of a medicament for treating oral tumors.
18. Use of a photocatalytic nanoenzyme according to any one of claims 1 to 6 as an antibacterial agent.
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