WO2022183811A1 - 一种仿过氧化氢酶活性的光催化纳米酶及其制备方法和应用 - Google Patents

一种仿过氧化氢酶活性的光催化纳米酶及其制备方法和应用 Download PDF

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WO2022183811A1
WO2022183811A1 PCT/CN2021/139346 CN2021139346W WO2022183811A1 WO 2022183811 A1 WO2022183811 A1 WO 2022183811A1 CN 2021139346 W CN2021139346 W CN 2021139346W WO 2022183811 A1 WO2022183811 A1 WO 2022183811A1
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photocatalytic
mop
nanozyme
nanoparticles
preparation
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李志斌
梅婷婷
程自强
喻学锋
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中国科学院深圳先进技术研究院
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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
    • B01J35/00—Catalysts, in general, characterised by their form or physical properties
    • B01J35/30—Catalysts, in general, characterised by their form or physical properties characterised by their physical properties
    • B01J35/39—Photocatalytic properties
    • 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
    • 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
    • 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
    • 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

Definitions

  • the invention belongs to the technical field of medical and health care, and in particular relates to a photocatalytic nano-enzyme imitating catalase activity and a preparation method and application thereof.
  • Hydrogen peroxidase also known as Catalase (CAT)
  • CAT Catalase
  • Nanozymes are a class of mimetic enzymes that have both the unique properties of nanomaterials and catalytic functions. Nanozymes not only have the high catalytic activity of natural enzymes, but also have the characteristics of stable and economical simulated enzymes. Therefore, since the report of HRP nanozymes in 2007, the research of nanozymes has risen rapidly, and the research scope has gradually expanded, including materials science. , physics, chemistry, biology, medicine and the environment. The emergence of nanozymes provides a new method for tumor diagnosis. The specific recognition and high catalytic activity of tumor cells using nanozymes can catalyze H 2 O 2 overexpressed in tumor lesions to generate strong oxidative active species such as hydroxyl radicals ( OH) to induce tumor cell apoptosis.
  • OH hydroxyl radicals
  • MoP 2 has unique properties such as small effective mass, high carrier mobility, and small energy band overlap. At present, it is mostly used in the field of photoelectric catalysis, and there is no report on the application of MoP 2 as a nanozyme in medical and health care.
  • MoP2 nanoparticles have good biocompatibility, photothermal conversion efficiency, and peroxide - like activity, and have strong H2O2 catalytic activity under near - infrared light irradiation, which can catalyze H2O2 to rapidly generate hydroxyl groups
  • Strong oxidative active species such as free radicals ( OH) can kill bacteria in the tumor microbial environment while inducing tumor cell apoptosis.
  • MoP2 nanoparticles can be gradually degraded into free metal ions and nontoxic phosphates. Since phosphorus is an essential element in the human body and exists in a large amount (1%) in the human body and molybdenum is a trace element required by the human body, the degradation products of MoP 2 nanoparticles can be absorbed or excreted by the human body, which has good biological safety. Therefore, the photocatalytic MoP 2 nanoparticles used in the present invention can be used in the medical and health field as a catalase-mimicking nano-enzyme.
  • the purpose of the present invention is to provide a preparation method and application of a photocatalytic nanozyme with catalase activity.
  • the preparation method of the invention is simple, and the prepared photocatalytic nanozyme has high H 2 O 2 catalytic activity, excellent stability and good biocompatibility. It can rapidly catalyze H 2 O 2 to generate hydroxyl radicals ( ⁇ OH) under light, which can lead to apoptosis of tumor cells by destroying lipids, nucleic acids, proteins and other biomolecular substances in cells.
  • ⁇ OH hydroxyl radicals
  • the high concentration of OH can rapidly decompose the enzymes, RNA, lysozyme and other substances contained in the bacteria, so as to achieve a better antibacterial effect.
  • the photocatalytic nanozyme has great application potential in medical and health fields such as tumor treatment, sterilization and disinfection.
  • the photocatalytic nanozyme was prepared from MoP 2 crystals.
  • the MoP2 crystals are formed by the interaction of red phosphorus and molybdenum through high temperature reaction in a vacuum-sealed environment followed by rapid cooling.
  • the mass ratio of the red phosphorus and molybdenum is 1.69 ⁇ 4:1.
  • the high temperature reaction temperature is between 600°C and 1000°C and lasts for 16 to 18 hours.
  • the cooling rate is between 1 ⁇ 3°C/min, and the temperature is cooled to 20 ⁇ 35°C.
  • the present invention also provides a method for preparing the above-mentioned photocatalytic nanozyme, comprising the following steps:
  • MoP2 nanoparticles were irradiated with near-infrared laser light to obtain photocatalytic nanozymes. Rapidly catalyzes H2O2 to generate reactive oxides such as hydroxyl radicals ( OH ) .
  • the solvent in step (1) is one of N-methyl-2-pyrrolidone, N,N-dimethylformamide, n-butanol or ethanol;
  • the ultrasonic treatment time in step (1) is 5-10 h;
  • the centrifugal speed in step (1) is 2000-4000 rpm, the centrifugation time is 10-20 min;
  • the secondary centrifugal speed in step (2) is 10,000 rpm, and the centrifugal time is 10-20 min;
  • the MoP 2 nanoparticles in step (2) are irregular in shape, the transverse diameter is between 100 and 500 nm, and the longitudinal diameter is between 50 and 200 nm.
  • the dispersion in step (2) can be water, ethanol, PBS buffer, 0.9% normal saline for injection;
  • the concentration of the MoP 2 nanoparticles dispersed in the dispersion liquid is 20-50 ⁇ g mL -1 .
  • the near-infrared laser wavelength in step (3) is 780 ⁇ 2526 nm.
  • the temperature of the MoP 2 nanoparticle dispersion liquid after the near-infrared laser irradiation in step (3) is ⁇ 45°C.
  • the photocatalytic nanozyme has extremely high H 2 O 2 catalytic efficiency, and can rapidly catalyze H 2 O 2 to generate active oxides such as hydroxyl radicals ( ⁇ OH).
  • the present invention also provides the application of the above-mentioned photocatalytic nanozyme in medical and health.
  • the photocatalytic nanozymes were administered in different doses, depending on the clinical status of cancer patients diagnosed early.
  • the photocatalytic nanozyme of the present invention has extremely strong stability in tumor lesions with complex microbial environment.
  • the H 2 O 2 catalytic activity can be excited in real time by near-infrared light, and the H 2 O 2 in the tumor lesion area can be rapidly catalyzed to generate reactive oxides such as hydroxyl radicals ( OH), which can effectively kill tumor cells.
  • the photocatalytic nanozyme has high catalytic activity under near-infrared light irradiation, and can rapidly catalyze active oxides such as hydroxyl radicals ( ⁇ OH) generated by H 2 O 2 , and can effectively and rapidly sterilize.
  • active oxides such as hydroxyl radicals ( ⁇ OH) generated by H 2 O 2 , and can effectively and rapidly sterilize.
  • the dosage is small and the sterilization efficiency is high.
  • the photocatalytic nanozyme of the present invention has high H 2 O 2 catalytic efficiency, solution stability, photodynamic stability, and high active oxygen production rate under infrared light irradiation, and has a very wide range of applications.
  • the photocatalytic nanozyme of the present invention has good biocompatibility. It can be gradually degraded into free molybdenum ions and non-toxic phosphates in the human body. Molybdenum is an essential trace element for the human body, and both molybdenum ions and phosphates are harmless to the human body.
  • the preparation method of the photocatalytic nano-enzyme of the present invention is simple, the catalytic efficiency is high, the dosage used is small, and the application in the medical and health fields such as tumor treatment and sterilization and disinfection is simple and effective.
  • Fig. 1 is the TEM image of MoP nanoparticle prepared in Example 1 of the present invention.
  • Fig. 2 is the activity diagram of the MoP 2 nanoparticles prepared in Example 1 of the present invention catalyzing the decomposition of H 2 O 2 under the induction of infrared laser;
  • Figure 3 is a diagram showing the efficiency of the MoP 2 nanoparticles prepared in Example 1 of the present invention catalyzing the generation of hydroxyl radicals ( OH) under infrared laser induction;
  • Fig. 4 is the cytotoxicity test chart of MoP 2 nanoparticles prepared in Example 1 of the present invention.
  • FIG. 5 is a comparison diagram of the anti-tumor cell performance of MoP 2 nanoparticles prepared in Example 1 of the present invention with or without infrared laser induction;
  • Fig. 6 is the test chart of the survival rate of Escherichia coli under the induction of infrared laser irradiation with or without the MoP 2 nanoparticles prepared in Example 1 of the present invention
  • Fig. 7 is the test chart of the survival rate of Staphylococcus aureus under the induction of infrared laser irradiation with or without the MoP 2 nanoparticles prepared in Example 1 of the present invention
  • Fig. 8 SEM morphology changes of Escherichia coli and Staphylococcus aureus of MoP 2 nanoparticles prepared in Example 1 of the present invention with or without infrared laser irradiation.
  • the MoP2 nanoparticles prepared in Example 1 were irregular in shape, with a transverse diameter ranging from 100 to 400 nm and a length of 80 to 100 nm. It has good H 2 O 2 catalytic activity and stability under the irradiation of infrared laser, and can efficiently catalyze H 2 O 2 to form active oxides such as hydroxyl radicals ( OH), which are used for tumor treatment or sterilization and other medical and health care application in the field.
  • active oxides such as hydroxyl radicals ( OH)
  • TEM Transmission electron microscopy
  • CAL27 xenogeneic oral tumor cells and SCC-9 human tongue squamous cell carcinoma cells were seeded in 96-well plates (about 1104 cells per well) and incubated overnight.
  • the specific operation is as follows: adding 100 ⁇ mol mL -1 of H 2 O 2 to each group, adding 40 ⁇ g mL -1 MoP 2 nanoparticles to groups b and d for incubation, and irradiating groups c and d with near infrared (0.5 W cm -2 ) after 10 minutes, add hydrogen peroxide detection reagent (Beyotime Biotech, Shanghai, China) to each group of samples according to the instructions for use, mix 50 ⁇ L of sample with 100 ⁇ L of reagent at room temperature for 30 min, and then use enzyme The absorbance was measured at 560 nm using a standard spectrometer (Multisken sky, ThermoFisher, China). The H 2 O 2 concentration ( ⁇ mol mL ⁇ 1 ) in each group was calculated using the standard curve of the standard solution.
  • CAL27 xenogeneic oral tumor cells and SCC-9 human tongue squamous cell carcinoma cells were seeded in 96-well plates (about 1104 cells per well) and incubated overnight.
  • the specific implementation operations are: adding 100 ⁇ mol mL -1 of H 2 O 2 to groups a, c, e, and f, adding 40 ⁇ g mL -1 MoP 2 nanoparticles to groups b, c, d, and f for incubation, After 10 minutes of near-infrared irradiation (0.5 W cm -2 ) in groups d, e, and f, the culture medium and trypsin-digested cells of each group were collected, mixed with 500 ⁇ mol mL -1 terephthalic acid (TA), The solution was placed in a 37°C orbital incubator and shaken gently in the dark for 12 hours, and then the change in the fluorescence emission peak at 435 nm was measured by fluorescence spectroscopy (F-4600, Hitachi, Japan). Percent enhancement is calculated as (Ftest Fblank)/(Fcontrol Fblank), where Fblank is the initial amount of fluorescence intensity in the presence of TA.
  • Fig. 3 shows that MoP 2 nanoparticles can generate a small amount of hydroxyl radicals in the H 2 O 2 environment, and MoP 2 nanoparticles activated by near-infrared light can generate a large number of hydroxyl radicals in the H 2 O 2 environment, It shows that the photocatalytic MoP 2 nanoparticles have strong catalase activity, which can quickly and efficiently catalyze the decomposition of H 2 O 2 to generate a large number of hydroxyl radicals ( ⁇ OH).
  • This example evaluates the cytotoxicity of the MoP 2 nanoparticles prepared by the present invention.
  • Xenogeneic oral tumor cells (CAL27), human tongue squamous cell carcinoma cells (SCC9), and human oral keratinocytes (HOK) were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO . Cultivated in a humidified atmosphere. The cytotoxicity of MoP 2 nanoparticles was evaluated using the standard cell counting kit-8 (CCK-8) method.
  • xenogeneic oral tumor cells (CAL27), human tongue squamous cell carcinoma cells (SCC9) and human oral keratinocytes (HOK) were placed in 48-well plates (2 ⁇ 10 4 cells/ (well), incubated at 37 °C and 5% CO for 24 h, respectively.
  • the medium was then replaced with 200 ⁇ L of new complete medium containing MoP 2 nanoparticles at various concentrations. After 24 hours of incubation, the medium was removed, washed three times with phosphate-buffered saline (PBS), and 10 ⁇ L of the cell counting kit was added.
  • CCK-8 reagent further cultured at 37 °C for 1.5 h.
  • This example evaluates the anti-tumor cell effect of the MoP 2 nanoparticles prepared by the present invention under near-infrared light irradiation.
  • Xenogeneic oral tumor cells (CAL27) and human tongue squamous cell carcinoma cells (SCC9) were cultured in DMEM medium containing 10% fetal bovine serum, respectively, at 37 °C and 5% CO2 in a humidified atmosphere.
  • the biocompatibility of nanospheres was tested by live/dead cell staining kit (Calcein AM/PI).
  • Calcein AM is an excellent fluorescent dye for living cells, which can easily penetrate the characteristics of living cells. It will be hydrolyzed into calcein by esterase and remain in the cell, showing strong green fluorescence; propidium iodide (PI) in the kit cannot pass through the living cell membrane, but can pass through the disordered area of the dead cell membrane to reach the nucleus. Red fluorescence (excitation wavelength of 535 nm, emission wavelength of 617 nm) is generated by the helix of cellular DNA, which makes dead cells appear red fluorescent.
  • a near-infrared laser (1W/cm 2 ) with a wavelength of 808 nm for 10 minutes or not at all.
  • the specific operation is as follows: take the xenogeneic oral tumor cells (CAL27) and human tongue squamous carcinoma cells (SCC9) in the logarithmic growth phase, respectively, at 5 ⁇ 10 4 /mL, 400 ⁇ L per well, and add them to a 48-well plate, and place them in a cell incubator. After culturing in medium for 24 hours, the cell culture medium in each well was gently aspirated, and 400 ⁇ L of cell culture medium containing different components to be tested was added to the well for 24 hours of continuous culture, with serum-free medium as a blank control.
  • CAL27 xenogeneic oral tumor cells
  • SCC9 human tongue squamous carcinoma cells
  • This example evaluates the antibacterial effect of the MoP 2 nanoparticles prepared by the present invention under near-infrared light irradiation.
  • Escherichia coli and Staphylococcus aureus were used as bacterial models, and the experimental groups were set as: a is blank control group; b is MoP 2 nanoparticle dispersion (40 ⁇ g mL -1 ); c is H 2 O 2 solution (100 ⁇ M) , d are MoP 2 nanoparticle dispersion (40 ⁇ g mL -1 ) and H 2 O 2 solution (100 ⁇ M). All experimental groups were irradiated with near-infrared laser (1W/cm 2 ) with a wavelength of 808 nm for 10 minutes or not at all.
  • the specific implementation operations are as follows: pick a small amount of bacterial preservation solution and add it to the bacterial culture medium containing LB liquid, place it in a shaking tube at 37 °C, shake overnight in a 240 rmp bacterial constant temperature incubator, and then use a refrigerated centrifuge (5000 rmp , 2 min) to collect the bacteria, wash the bacteria with sterile saline, adjust the bacterial concentration with saline, and use a multi-function microplate reader for detection, so that the concentration of the bacterial suspension reaches an OD600 of 0.01 (ie (0.4- 0.5) x 10 6 cfu/mL).
  • the morphological changes of Escherichia coli and Staphylococcus aureus after different treatments were observed by scanning electron microscope (SEM).
  • SEM scanning electron microscope
  • the specific real-time operation was as follows: the bacterial precipitate was dispersed in a 2.5% glutaraldehyde solution and fixed overnight at 4°C. The fixed bacteria were washed three times with PBS, dehydrated with 25, 50, 80, and 100 wt% ethanol series for 10 min in turn, and completely dried at room temperature. Then, the bacteria were sputtered with gold (30 s, 30 mA) and observed with a Zeiss Sigma 300 scanning electron microscope.
  • Fig. 8 shows that MoP 2 nanoparticles in H 2 O 2 environment can distort the morphology and shrink the membrane structure of Escherichia coli and Staphylococcus aureus under near-infrared light, while the two nanoparticles in the absence of near-infrared light The inoculum remains in its original form.
  • the present invention illustrates the preparation and application scheme of a catalase-mimicking photocatalytic nano-enzyme of the present invention through the above examples.
  • the present invention is not limited to the above-mentioned embodiments, and the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Abstract

本发明涉及医疗卫生领域，具体涉及一种仿过氧化氢酶活性的光催化纳米酶以及制备方法和应用。所述光催化纳米酶由MoP 2晶体制备而得，所制备的MoP 2纳米颗粒在近红外激光照射处理后得到具有H 2O 2催化活性的光催化纳米酶，可催化H 2O 2迅速产生大量羟基自由基（·OH）等活性自由基。相比于现有的过氧化氢酶，红外激光介导的MoP 2纳米颗粒可在不使用过氧化氢酶的复杂微环境中高效催化H 2O 2，在肿瘤治疗、作为抗菌剂以及杀菌消毒等医疗卫生领域具有广泛的应用。

Description

一种仿过氧化氢酶活性的光催化纳米酶及其制备方法和应用 技术领域
本发明属于医疗卫生技术领域，具体涉及一种仿过氧化氢酶活性的光催化纳米酶及其制备方法和应用。
 背景技术
过氧化氢酶（Hydrogen peroxidase）又称触酶（Catalase, CAT），是一类广泛存在于动物、植物和微生物体内的末端氧化酶，是以过氧化氢为底物，通过催化一对电子的转移而最终将其分解为水和氧气。过氧化氢酶在食品、医药、纺织、造纸、环保等行业具有重要的应用。当过氧化氢酶应用于有机组织的处理时，其活性受到温度、pH值、H 2O 2含量等微环境的影响，限制了其在医疗卫生领域的应用。
纳米酶是一类既有纳米材料的独特性能，又有催化功能的模拟酶。纳米酶既有天然酶的高催化活性，又有模拟酶稳定而经济的特点，因此自2007年HRP纳米酶报道以来，纳米酶的研究迅速崛起，研究的涉及面也逐渐广泛，已经包括材料科学、物理、化学、生物、医学和环境等不同领域。纳米酶的出现为肿瘤的诊断提供了新的方法。利用纳米酶的肿瘤细胞的特异性识别与高催化活性，催化肿瘤病灶区过表达的H 2O 2产生羟基自由基（·OH）等强氧化性活性物种，以诱导肿瘤细胞凋亡。由于一定浓度的·OH能破坏细菌的生物大分子，包括DNA、细胞蛋白质和膜脂等，起到杀菌的作用。因此在肿瘤治疗上具有较为广阔的应用前景。然而，纳米酶在复杂的肿瘤微环境中的催化效率受到限制，使得产生的·OH自由基量极少而影响治疗效果。而要满足·OH自由基治疗肿瘤需要的产生量，需要增大纳米酶的剂量，这会导致一些不可抑制的副作用，如肾毒性和严重过敏反应等。因此，迫切需要发现在复杂微生物环境中具有高催化活性的纳米酶。
MoP 2具有有效质量小、载流子迁移率高、能带重叠能小等独特性能。目前多用于光电催化领域的研究，尚未见MoP 2作为纳米酶应用于医疗卫生的报道。MoP 2纳米颗粒具有良好的生物相容性、光热转换效率及类过氧化物的活性，在近红外光照射下具有极强的H 2O 2催化活性，能够催化H 2O 2快速产生羟基自由基（·OH）等强氧化性活性物种，在诱导肿瘤细胞凋亡的同时杀灭肿瘤微生物环境中的细菌。在复杂的体内环境中，MoP 2纳米颗粒可以逐渐降解为游离金属离子和无毒的磷酸盐。由于磷是人体必需元素，在人体大量存在（1%）而钼是人体所需的微量元素，MoP 2纳米颗粒的降解产物能被人体吸收或排出，具有较好的生物安全性。因此，本发明所使用的光催化MoP 2纳米颗粒可以作为仿过氧化氢酶的纳米酶应用在医疗卫生领域中。
技术问题
针对现有技术的不足，本发明的目的在于提供一种具有过氧化氢酶活性的光催化纳米酶的制备方法和应用。本发明制备方法简单，且制得的光催化纳米酶具有较高的H 2O 2催化活性，极好的稳定性，还表现出很好的生物相容性。在光照下能迅速催化H 2O 2产生羟基自由基(·OH)，可以通过破坏细胞的脂类、核酸、蛋白质等生物分子物质，导致肿瘤细胞的凋亡。同时，高浓度的·OH能使细菌内含的酶素、RNA、溶菌酶等物质迅速分解，从而达到较好的抗菌效果。所述光催化纳米酶在的肿瘤治疗、杀菌消毒等医疗卫生领域具有巨大的应用潜力。
技术解决方案
为达到此发明目的，本发明采用以下技术方案：
一种具有过氧化氢酶活性的光催化纳米酶的制备方法和应用。所述光催化纳米酶由MoP 2晶体制备而得。所述MoP 2晶体由红磷和钼通过在真空密封环境中高温反应然后迅速冷却相互作用而成。所述红磷和钼的质量比为1.69~4:1。
进一步地，所述高温反应温度在600℃~1000℃之间，持续16~18 h。
进一步地，所述冷却速度在1~3℃/min之间，冷却至20~35℃。
本发明还提供上述光催化纳米酶的制备方法，包括如下步骤：
（1）将上述MoP 2晶体在溶剂中超声处理后离心，收集上清液；
（2）将上清液二次离心，取沉淀物冲洗数次，得到MoP 2纳米颗粒，重新悬浮于分散液中；
（3）用近红外激光照射MoP 2纳米颗粒，获得光催化纳米酶。快速催化H 2O 2产生羟基自由基（·OH）等活性氧化物。
进一步地，所述制备方法中，步骤（1）中的溶剂为N -甲基-2-吡咯烷酮、N,N-二甲基甲酰胺、正丁醇或乙醇中的一种；
进一步地，所述制备方法中，步骤（1）中的超声处理时间为5~10 h；
进一步地，所述制备方法中，步骤（1）中的离心速度为2000~4000 rpm，离心时间为10~20 min;
进一步地，所述制备方法中，步骤（2）中的二次离心速度为10000rpm，离心时间为10~20 min;
进一步地，所述制备方法中，步骤（2）中的MoP 2纳米颗粒呈不规则形状，横向直径在100~500 nm之间，纵向直径在50~200 nm之间。
进一步地，所述制备方法中，步骤（2）中的分散液可以是水、乙醇、PBS缓冲液、0.9%注射用生理盐水；
进一步地，所述制备方法中，步骤（2）中分散液分散MoP 2纳米颗粒浓度为20~50 μg mL -1。
进一步地，所述制备方法中，步骤（3）中的近红外激光波长为780~ 2526 nm。
进一步地，所述制备方法中，步骤（3）中的近红外激光照射后MoP 2纳米颗粒分散液的温度≤45℃。
进一步地，所述光催化纳米酶具有极高的H 2O 2催化效率，能快速催化H 2O 2产生羟基自由基（·OH）等活性氧化物。
本发明还提供上述光催化纳米酶在医疗卫生上的应用。
进一步地，所述应用于肿瘤治疗中的应用。
所述光催化纳米酶通过不同剂量的给药，这取决于早起诊断的癌症患者临床状况。本发明的光催化纳米酶在具有复杂微生物环境的肿瘤病灶区具备极强的稳定性。通过近红外光照能实时激发其H 2O 2催化活性，迅速催化肿瘤病灶区的H 2O 2产生羟基自由基（·OH）等活性氧化物，有效杀死肿瘤细胞。
进一步地，所述应用于杀菌消毒中的应用。
所述光催化纳米酶在近红外光照射下催化活性高，迅速催化H 2O 2产生的羟基自由基（·OH）等活性氧化物能有效、快速杀菌消毒。使用剂量小，杀菌效率高。
有益效果
本发明具有以下技术特点：
（1）本发明所述光催化纳米酶在红外光照射下具有很高的H 2O 2催化效率，溶液稳定性，光动力稳定性，以及高活性氧产氧率，具备非常广泛的应用。
（2）本发明所述光催化纳米酶具有较好的生物相容性。其在人体中可以逐渐降解为游离钼离子和无毒的磷酸盐，钼元素为人体必需的微量元素，钼离子与磷酸盐均对于人体无害。
（3）本发明所述的光催化纳米酶制备方法简单，催化效率高，使用剂量小，在肿瘤治疗与杀菌消毒等医疗卫生领域的应用简单、有效。
 附图说明
图1为本发明实施例1制备得到的MoP 2纳米颗粒的透射电镜图；
图2 为本发明实施例1制备得到的MoP 2纳米颗粒在红外激光诱导下催化H 2O 2分解的活性图；
图3 为本发明实施例1制备得到的MoP 2纳米颗粒在红外激光诱导下催化产生羟基自由基（·OH）的效率图；
图4为本发明实施例1制备得到的MoP 2纳米颗粒的细胞毒性测试图；
图5为本发明实施例1制备得到的MoP 2纳米颗粒有无红外激光诱导下的抗肿瘤细胞性能对比图；
图6为本发明实施例1制备得到的MoP 2纳米颗粒有无红外激光照诱导下的大肠杆菌存活率测试图；
图7为本发明实施例1制备得到的MoP 2纳米颗粒有无红外激光照诱导下的金黄色葡萄球菌存活率测试图；
图8 本发明实施例1制备得到的MoP 2纳米颗粒有无红外激光照诱导下的大肠杆菌和金黄色葡萄球菌SEM形貌变化图。
本发明的最佳实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1：
用天平称取100 mg钼粉与300 mg红磷块，至于石英管的一端，真空密封。将石英管水平放置在马弗炉中，以850℃的温度加热18小时高温反应，然后以2℃/min的速度冷却至30℃。取出石英管热端形成的MoP 2晶体，在N-甲基-2-吡咯烷酮(NMP)中超声处理10 h后，以4000 rpm的速度离心10 min，得到MoP 2纳米颗粒。收集含有MoP 2纳米颗粒的上清液在10000 rpm的转速下离心15分钟。将所得沉淀物用乙醇冲洗两次后重新悬浮于PBS缓冲液中备用。
实施例2：
实施例1制备的MoP 2纳米颗粒呈不规则形状，横向直径在100~400 nm之间，长度80~100 nm。在红外激光的照射下具有很好的H 2O 2催化活性和稳定性，可高效催化H 2O 2形成羟基自由基（·OH）等活性氧化物，用于肿瘤治疗或杀菌消毒等医疗卫生领域的应用。
使用透射电子显微镜（TEM）观察实施例1制备的MoP 2纳米颗粒形貌与纳米尺寸，并对其在红外光照下的H 2O 2催化活性进行表征，监测溶液中羟基自由基(·OH)的形成情况。具体测试结果如下：
（1）透射电子显微镜（TEM）
透射电子显微镜（TEM）表示所制备的MoP 2纳米颗粒的形貌和纳米尺寸。结果参照图1，表示在200nm的标尺下，检测MoP 2纳米颗粒的分布及形貌，从图1中可以说明MoP 2纳米颗粒呈现不规则的棒状结构。
（2）H 2O 2催化效率
将CAL27异种口腔肿瘤细胞和SCC-9人舌鳞癌细胞接种于96孔板（每孔约1104细胞），孵育过夜。设置细胞实验组：a为对照组（H 2O 2）、b为MoP 2纳米颗粒催化组（ H 2O 2 + MoP 2）、c为红外光催化组（H 2O 2 + NIR）、d为红外光介导MoP 2纳米颗粒催化组（H 2O 2 + MoP 2 + NIR）。
具体实施操作为：每组中加入100 μmol mL -1的 H 2O 2，在b、d组中加入 40 μg mL -1 MoP 2纳米颗粒孵导，对c、d组进行近红外照射(0.5 W cm -2) 10分钟后，按照使用说明在各组样品中加入过氧化氢检测试剂(中国上海Beyotime Biotech)，将50 μL的样品与100 μL的试剂在室温下混合30 min，然后用酶标仪(Multisken sky, ThermoFisher, China)在560 nm处测定吸光度。用标准溶液的标准曲线计算各组中H 2O 2浓度(μmol mL -1)。
结果参照图2，表示与对照组相比，在近红外光照射MoP 2纳米颗粒的条件下H 2O 2的含量急剧下降。而单独使用MoP 2纳米颗粒或单独使用近红外光照射对H 2O 2含量没有太大的影响。表示经近红外光激活的MoP 2纳米颗粒有很强的催化活性，能有效催化H 2O 2的分解。
（3）羟基自由基（·OH）的产生效率
将CAL27异种口腔肿瘤细胞和SCC-9人舌鳞癌细胞接种于96孔板（每孔约1104细胞），孵育过夜。设置细胞实验组：a为H 2O 2、b为MoP 2纳米颗粒、c为MoP 2纳米颗粒和H 2O 2（MoP 2 + H 2O 2）、d为近红外光催化MoP 2纳米颗粒（MoP 2 + NIR）、e为近红外光催化H 2O 2（H 2O 2 + NIR）、f为H 2O 2环境中红外光催化MoP 2纳米颗粒（MoP 2 + H 2O 2 + NIR）。
具体实施操作为：向a、c、e、f组中加入100 μmol mL -1 of H 2O 2，b、c、d、f组中加入40 μg mL -1 MoP 2纳米颗粒孵导，对d、e、f组进行近红外照射(0.5 W cm -2) 10分钟后，收集各组的培养基和胰蛋白酶消化的细胞，与500 μmol mL -1对苯二甲酸(TA)混合，将溶液置于37℃轨道培养箱中，在黑暗中轻轻震荡12小时，然后用荧光光谱法(F-4600，日立，日本)测量435 nm处荧光发射峰的变化。增强百分数按(Ftest Fblank)/(Fcontrol Fblank)计算，其中Fblank为TA存在时荧光强度的初始量。
结果参照图3，表示MoP 2纳米颗粒在H 2O 2环境中能产生少量的羟基自由基，经近红外光激活的MoP 2纳米颗粒在H 2O 2环境中能产生大量的羟基自由基，说明光催化的MoP 2纳米颗粒有极强的过氧化氢酶活性，能迅速高效催化H 2O 2分解产生大量的羟基自由基（·OH）。
实施例3：
本实施例对本发明制备的MoP 2纳米颗粒的细胞毒性进行评价。
将异种口腔肿瘤细胞（CAL27）、人舌鳞癌细胞（SCC9）和人口腔角质形成细胞（HOK）分别培养于含10%胎牛血清的DMEM培养基中，在37℃和5% CO 2的湿化气氛中培养。采用标准细胞计数试剂盒-8 (CCK-8)法评价MoP 2纳米颗粒的细胞毒性。
具体实施操作为：将异种口腔肿瘤细胞（CAL27）、人舌鳞癌细胞（SCC9）和人口腔角质形成细胞（HOK）分别用400μL完全培养基置于48孔板(2×10 4个细胞/孔)上，分别在37℃、5% CO 2下孵育24 h。然后，介质被替换为200 μL新完全培养基含有不同浓度的MoP 2纳米颗粒，培养24小时之后，撤掉培养基，用磷酸盐缓冲盐水洗(PBS)三次，再加入10 μL 细胞计数试剂盒CCK-8 试剂，进一步培养在37 ℃中培养1.5 h。随后，用酶标仪(Varioskan Flash 4.00.53, Finland)测定活细胞在450 nm处的光密度(OD)。细胞存活率(%)= (ODtest -ODblank)/(ODcontrol-ODblank) 100%。
结果参照图4，表示不同浓度的MoP 2纳米颗粒的加入对异种口腔肿瘤细胞（CAL27）、人舌鳞癌细胞（SCC9）和人口腔角质形成细胞（HOK）均具有较好的细胞相容性，细胞存活率较高。
实施例4：
本实施例对本发明制备的MoP 2纳米颗粒在近红外光照射下的抗肿瘤细胞效果进行评价。
将异种口腔肿瘤细胞（CAL27）、人舌鳞癌细胞（SCC9）分别培养于含10%胎牛血清的DMEM培养基中，在37℃和5% CO 2的湿化气氛中培养。通过活/死细胞染色试剂盒（Calcein AM/PI）来检测纳米球的生物相容性，利用Calcein AM为活细胞优良的荧光染色剂，其能轻易穿透活细胞的特点，当其到达细胞内会被酯酶水解为钙黄绿素留在胞内，呈现强绿色荧光；试剂盒中的碘化丙啶（PI）不能穿过活细胞膜，但能穿过死细胞膜的无序区域到达细胞核，在嵌入细胞DNA螺旋后产生红色荧光（激发波长为535 nm，发射波长为617 nm）从而使死细胞呈现红色荧光。
设置细胞实验组：a为空白对照组；b为MoP 2纳米颗粒分散液（40 μg mL -1）；c为H 2O 2溶液（100 μM），d为MoP 2纳米颗粒分散液（40 μg mL -1）和H 2O 2溶液（100μM）。所有实验组均用波长为808nm的近红外激光（1W/cm 2）照射10分钟或均不照，处理完后进行细胞活死表征。
具体实施操作为：分别取对数生长期的异种口腔肿瘤细胞（CAL27）与人舌鳞癌细胞（SCC9）以5×10 4/mL，每孔400 μL加入48孔板内，于细胞培养箱中培养24 h后轻轻吸去每孔中的细胞培养基，将含有不同待测组分的细胞培养基400 μL加入孔中继续培养24 h，以无血清培养基作为空白对照。取出48孔细胞培养板，吸除孔内培养基，用PBS溶液小心清洗孔板3次后向每孔内加入钙黄绿素和碘化丙啶溶液，在培养箱中孵育20分钟后，吸除染料并用PBS溶液小心清洗3次后置于荧光显微镜下进行观察计数。
结果参照图5，表示MoP 2纳米颗粒在近红外光照下能够有效诱导异种口腔肿瘤细胞（CAL27）与人舌鳞癌细胞（SCC9）凋亡。而在没有近红外光照的情况下具有很好的生物相容性。
实施例5：
本实施例对本发明制备的MoP 2纳米颗粒在近红外光照射下的抗菌效果进行评价。
使用大肠杆菌与金黄色葡萄球菌为细菌模型，设置实验组为：a为空白对照组；b为MoP 2纳米颗粒分散液（40 μg mL -1）；c为H 2O 2溶液（100 μM），d为MoP 2纳米颗粒分散液（40 μg mL -1）和H 2O 2溶液（100 μM）。所有实验组均用波长为808 nm的近红外激光（1W/cm 2）照射10分钟或均不照。
（1）平板计数法
具体实施操作为：挑取少量细菌保菌液加到含有LB液体的细菌培养基中，置于摇菌管内于37℃，240 rmp细菌恒温培养箱中振荡过夜培养，随后用冷冻离心机（5000 rmp，2 min）对细菌进行收集，并使用无菌生理盐水洗涤细菌后，用生理盐水调节细菌浓度，用多功能酶标仪进行检测，使细菌悬液的浓度达到OD600为0.01（即（0.4-0.5）×10 6 cfu/mL）。
将100 µL上述各样品与100 µL活化的菌悬液共混后均匀涂布于培养皿表面，菌液均匀涂布于培养皿后，将培养皿置于细菌培养箱中37℃恒温恒湿培养18 h，观察菌落个数。光照组用808 nm（1 W/cm 2）近红外光激光器光照10 min后，菌液均匀涂布于培养皿后，将培养皿置于细菌培养箱中37℃恒温恒湿培养18 h，观察菌落个数。每个样品平行重复5次。空白对照组将样品溶液换为等体积的生理盐水。
结果参照图6、图7，表示H 2O 2环境中的MoP 2纳米颗粒在近红外光照下能有效消杀大肠杆菌和金黄色葡萄球菌，而在没有近红外光照的条件下对细菌没有太大的消杀作用。
（2）细菌SEM形态
通过扫描电镜（SEM）观察不同处理后大肠杆菌和金黄色葡萄球菌的形态变化。具体实时操作为：将细菌沉淀分散于2.5%戊二醛溶液中，4℃固定过夜。固定菌用PBS洗涤3次，依次用25、50、80、100 wt%的乙醇系列脱水10 min，室温下完全干燥。然后，将细菌溅射金(30 s, 30 mA)，用Zeiss Sigma 300扫描电镜观察。
结果参照图8，表示H 2O 2环境中的MoP 2纳米颗粒在近红外光照下能使大肠杆菌和金黄色葡萄球菌形态扭曲、膜结构皱缩明显，而在没有近红外光照条件下的两种菌保持原来的形态。
申请人声明，本发明通过上述实施例来说明本发明的一种仿过氧化氢酶的光催化纳米酶制备及其应用方案。但本发明并不局限于上述实施例，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (10)

  1. 一种仿过氧化氢酶的光催化纳米酶，其特征在于：所述光催化纳米酶由MoP 2晶体制备而得，所制备的MoP 2纳米颗粒在近红外激光照射处理后得到具有H 2O 2催化活性的光催化纳米酶。
  2. 根据权利要求1所述的仿过氧化氢酶的光催化纳米酶，其特征在于：所述MoP 2晶体由红磷和钼通过在真空密封环境中高温反应然后迅速冷却相互作用而成；
    优选的，所述红磷和钼的质量比为(1.69~4):1；
    优选的，所述高温反应温度为600℃~1000℃，反应时间为16~18h；
    优选的，所述冷却速度为1~3℃/min，冷却至20~35℃。
  3. 根据权利要求1或2所述仿过氧化氢酶的光催化纳米酶，其特征在于：所述光催化纳米酶呈不规则形状。
  4. 根据权利要求1或2所述的一种仿过氧化氢酶的光催化纳米酶的制备方法，其特征在于：包括如下步骤：
    （1）将MoP 2晶体在溶剂中超声处理后离心，收集上清液；
    （2）将上清液二次离心，取沉淀物冲洗数次，得到MoP 2纳米颗粒，重新悬浮于分散液中；
    （3）用近红外激光照射MoP 2纳米颗粒，获得具有H 2O 2催化活性的光催化纳米酶。
  5. 根据权利要求4所述的制备方法，其特征在于，步骤（1）中，所述溶剂为N -甲基-2-吡咯烷酮、N,N-二甲基甲酰胺、正丁醇或乙醇中的一种；超声处理时间为5~10 h，离心速度为2000~4000 rpm，离心时间为10~20 min。
  6. 根据权利要求4所述的制备方法，其特征在于，步骤（2）中，二次离心速度为10000 rpm，离心时间为10~20 min；所述分散液选自水、乙醇、PBS缓冲液、0.9%注射用生理盐水中的一种，优选的，所述分散液中MoP 2纳米颗粒的浓度为20~50μg mL -1。
  7. 根据权利要求4所述的制备方法，其特征在于，步骤（3）中，近红外激光波长为780~ 2526 nm，优选为780~1100 nm；
    优选的，步骤（3）中，近红外激光照射时间为10~30 min；
    优选的，步骤（3）红外激光照射后的光催化纳米酶分散液温度≤45℃。
  8. 一种抗菌材料，包括光催化纳米酶，所述光催化纳米酶由MoP 2晶体制备而得，所制备的MoP 2纳米颗粒在近红外激光照射处理后得到具有抗菌性能的光催化纳米酶。
  9. 根据权利要求1-3任一项所述的光催化纳米酶在制备肿瘤药物中的应用，优选的，所述光催化纳米酶在制备口腔肿瘤药物中的应用。
  10. 根据权利要求1-3任一项所述的光催化纳米酶作为抗菌剂的用途。
PCT/CN2021/139346 2021-03-05 2021-12-17 一种仿过氧化氢酶活性的光催化纳米酶及其制备方法和应用 WO2022183811A1 (zh)

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