CN113952984B - High-catalytic-activity molybdenum-based nanoenzyme and preparation method and application thereof - Google Patents

Abstract

The invention discloses a molybdenum-based nano-enzyme with high catalytic activity and a preparation method and application thereof, belonging to the technical field of nano-enzymes, which solves the problem of poor catalytic effect of molybdenum oxide-like catalase in the prior art, and also It solves the problem that the biological toxicity of copper ions is high, and the use of excessive doses will cause great side effects to the human body. The highly catalytically active molybdenum-based nanozyme of the present invention contains molybdenum oxide nanoparticles and copper ions, and the copper ions are anchored on the surface of the molybdenum oxide nanoparticles; the preparation materials of the highly catalytically active molybdenum-based nanozyme include molybdenum oxide nanoparticles, cysteine amino acids, copper ions and surfactants. The high catalytic activity molybdenum-based nanozyme of the present invention has good catalytic activity and radiotherapy sensitization function, has no obvious cytotoxicity, and is safe to use.

Description

A kind of high catalytic activity molybdenum-based nanozyme and its preparation method and application

technical field

The invention relates to the technical field of nano-enzymes, in particular to a high catalytic activity molybdenum-based nano-enzyme and a preparation method and application thereof.

Background technique

Inorganic nanomaterials, especially transition metal oxides, have special physicochemical properties, which have attracted intensive research due to their simple preparation, low price, and tunable local plasmon resonance phenomena similar to those of noble metals. Molybdenum oxide is a typical transition metal-semiconductor inorganic nanomaterial. Due to its excellent electrochromic, photochromic, catalytic activity, and electrode intercalation properties, it is widely used in the fields of color developers, photocatalysis, batteries, and gas sensors. Wide range of applications. In recent years, with the development of nanotechnology and nanomedicine, it has been found that nano-molybdenum oxide has the characteristics of low biological toxicity, easy surface modification, more oxygen holes, and strong near-infrared photothermal conversion ability. Medical fields such as antibacterial, antitumor, biomolecule detection, biocatalysis, etc. show broad application prospects.

Tumor micro-environment (TME)-mediated nanocatalytic therapy mainly uses non-toxic or low-toxic nanomaterials to selectively catalyze and trigger specific chemical reactions within the TME to locally generate a considerable amount of specific molecules. The tumor treatment strategy of reaction products such as reactive oxygen species (ROS) to achieve a range of biological and pathological responses and reduce side effects on normal tissues is a tumor-specific treatment modality. However, the efficiency of TME _- mediated nanocatalytic therapy is generally limited by intratumor _H2O2 levels, insufficient acidity, and hypoxia. Therefore, it has become one of the current research hotspots to enhance the effect of tumor catalytic therapy by regulating TME. The development of inorganic nanozymes (such as peroxidase peroxidase (POD), oxidase oxidase (OXD), catalase (CAT), etc.) with potential high-efficiency enzyme-like catalytic activity provides a great opportunity for tumor catalytic therapy. New opportunity. For example, CAT-like nanozymes can catalyze the decomposition of H ₂ O ₂ (concentration range: 100 μM-1.0 mM) highly expressed in TME into oxygen, thereby alleviating intratumoral hypoxia. However, the construction of highly catalytically active inorganic nanozymes for precise TME response and low toxicity to normal tissues to achieve efficient tumor suppression still faces great challenges.

Copper is a trace metal element in the human body and plays an important role in maintaining the health of the human cardiovascular system. Copper ions have been shown to have excellent properties to stimulate angiogenesis, collagen deposition processes to promote wound healing; in addition, some new properties of copper compounds or ions have been discovered and used in the biomedical field. For example, Cu-based polyoxometalate catalysts (Cu-MOFs) have good biocompatibility and excellent catalytic properties and can be used for drug delivery and tumor therapy (e.g., Cu ²⁺ radiosensitization, photothermal therapy, etc. ). However, due to the relatively high biological toxicity of copper ions, the use of excessive doses will cause great side effects to the human body.

SUMMARY OF THE INVENTION

In view of the above analysis, the present invention aims to provide a platinum-based nanozyme with high catalytic activity and a preparation method and application thereof, which have less toxic and side effects and high catalytic activity, and can at least solve one of the following technical problems: (1) separate oxidation The catalytic effect of molybdenum-like catalase is poor; (2) the biological toxicity of copper ions is relatively high, and the use of excessive doses in the anti-tumor process will cause great side effects to the human body.

The object of the present invention is mainly achieved through the following technical solutions:

In one aspect, the present invention provides a highly catalytically active molybdenum-based nanozyme, comprising molybdenum oxide nanoparticles and copper ions, and the copper ions are anchored on the surface of the molybdenum oxide nanoparticles.

Further, the raw materials for the preparation of the highly catalytically active molybdenum-based nanozyme include molybdenum oxide nanoparticles, cysteine, copper ions and surfactants.

Further, the surfactant is a polymer with good biocompatibility.

On the other hand, the present invention provides a kind of preparation method of high catalytic activity platinum-based nanozyme, comprising the following steps:

Step S1, preparing molybdenum oxide nanoparticles;

Step S2, adding cysteine and water-soluble copper salt to the secondary water and chelating it into a white floc L-Cys-Cu by ultrasonic stirring;

Step S3, placing the mixed solution of molybdenum oxide nanoparticles and L-Cys-Cu in a centrifuge tube, sonicating in an ultrasonic tank, then stirring on a magnetic stirrer, and adding surfactant uniformly and slowly during the stirring process;

Step S4: After the reaction, the precipitate is taken out by centrifugation, and the precipitate is repeatedly centrifuged and washed with secondary water for 2-3 times, and then washed with ethanol and centrifuged to absorb excess ethanol to obtain a platinum-based nanozyme with high catalytic activity.

Further, in the step S1, the step of preparing molybdenum oxide nanoparticles includes:

S101, weighing ammonium molybdate powder in proportion, dissolving the ammonium molybdate powder in deionized water, and ultrasonically dissolving to obtain an aqueous solution of ammonium molybdate;

S102, using a pipette gun to add absolute ethanol to the ammonium molybdate aqueous solution obtained in S101, ultrasonically dispersing and stirring at room temperature to obtain a uniformly mixed mixed solution;

S103, transfer the mixed solution into a hydrothermal reaction kettle, place the reaction kettle in an oven after tightening it, set the reaction temperature to be 160-200°C, and the reaction time to be 10-15h;

S104, after the reaction is completed, after the reaction is naturally cooled to room temperature, the precipitate is taken out by centrifugation, and the precipitate is repeatedly centrifuged and washed with secondary water for 2-5 times, and placed in a freeze dryer to freeze dry to obtain molybdenum oxide nanoparticles.

Further, it is characterized in that, in S101 and S102, ammonium molybdate: absolute ethanol: deionized water=0.7-1 mmol: 10-15 mL: 20-25 mL.

Further, in the step S2, the water-soluble copper salt is copper chloride, and the molar ratio of cysteine and copper chloride is 2:1-1.5.

Further, in the step S3, the molar ratio of the molybdenum oxide nanoparticles to the L-Cys-Cu is 1:1-3.

Further, in the step S3, the surfactant is polyvinylpyrrolidone, and the mass ratio of molybdenum oxide nanoparticles to polyvinylpyrrolidone is 1:1-2.

The present invention also provides the application of a high catalytic activity molybdenum-based nanozyme, which is used as an anti-tumor material.

Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

(1) The present invention utilizes a simple one-step hydrothermal method, decomposes ammonium molybdate under hydrothermal conditions and re _- nucleates and grows into porous MoO2 nanoparticles, which have high catalase-like catalytic activity, and can be used in catalysis It has degradation behavior during the process and can avoid the long-term toxicity of nanomaterials to organisms. This is a safe and efficient H2O2 _- responsive _nanoenzyme catalyst in TME, and can improve intratumor hypoxia and sensitize radiotherapy; in MoO ₂ In the process of anchoring copper ions on the surface of nanoparticles, there is only simple and safe ultrasonic stirring at room temperature, and no other high temperature and high pressure processes to change the morphology and structure of MoO ₂ nanoparticles, and the cysteine L-Cys acts as a The intermediate linking agent is used for ion anchoring, which provides a reference for other transition metal oxides to anchor metal ions with a simple synthetic idea, and the preparation method is simple and safe.

(2) The catalase-like catalytic activity and radiosensitization function of the highly catalytically active molybdenum-based nanozyme MoO ₂ -L-Cys-Cu-PVP (referred to as MCCP) of the present invention are much higher than those of the single molybdenum oxide and the single of L-Cys _- Cu, and H2O2 used in an amount of 100 μM _- 1.0 mM, in the high _- expressing dose range of H2O2 in the TME, in improving MCCP to relatively low concentrations of _{H2O2 -} like _hydrogen peroxide At the same time of the enzyme catalytic ability, the dosage of copper ions and H ₂ O ₂ with relatively high biological toxicity are also reduced, and finally a platinum-based nanozyme with excellent catalase-like mimetic catalytic activity is obtained. After MCCP catalyzes H ₂ O ₂ to generate oxygen, which is easily disassembled in TME under X-ray irradiation during radiotherapy, L-Cys-Cu is released from the composite and begins to undergo a Fenton-like reaction, resulting in more Therefore, MCCP improves tumor hypoxia and also improves tumor sensitivity during radiotherapy, and has synergistically enhanced catalase-like catalyzed decomposition of H ₂ O ₂ to generate oxygen to improve intratumor hypoxia and increase in tumor cells. Therefore, it has broad application prospects in the radiosensitization of tumors.

(3) The high catalytic activity platinum-based nanozyme of the present invention has no obvious cytotoxicity within the tested dose range, is safe to use, has significant effect, and has a wide application range.

In the present invention, the above technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be set forth in the description that follows, or will be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the description particularly pointed out in the written description and appended drawings.

Description of drawings

The drawings are for the purpose of illustrating specific embodiments only, and are not considered to be limitations of the present invention, and the same reference signs represent the same meanings throughout the drawings.

In Fig. 1, a is the scanning electron microscope figure of MCCP of the present invention; B is the transmission electron microscope figure of MCCP of the present invention; C is the element energy spectrum figure of MCCP; D is the X-ray powder diffraction figure of MCCP;

In Figure 2, a is the dissolved oxygen test results of MP nanoparticles with different concentrations; b is the consumption curve of MCCP to hydrogen peroxide; c is the catalase-like catalytic activity of the three materials MP, MCCP, and L-Cys-Cu. Comparison of the effect of H ₂ O ₂ ; d is the effect of temperature on the catalytic effect of MCCP;

Figure 3 is a Confocal Laser Scanning Microscopy (CLSM) image of MP and MCCP in the detection of reactive oxygen species (ROS) at the cellular level;

Figure 4 is a confocal laser scanning micrograph of an in vitro DNA double-strand break assay;

In Figure 5, a is the consumption curve of GSH with different concentrations of MCCP nanoparticles at the same time; b is the control experiment of MP, PBS and MCCP; c is the consumption curve of GSH with time under the same concentration of MCCP nanoparticles; d is X -Fluorescence emission spectrum of the product OH in the Fenton-like reaction between copper ions and H ₂ O ₂ under ray irradiation;

Figure 6 is the cytotoxicity data of MCCP nanoparticles and human umbilical vein epithelial cells HUVEC co-incubated for 24 hours;

Figure 7 shows the reaction mechanism of MCCP catalyzed H ₂ O ₂ to generate hydroxyl radicals in vitro detected.

Detailed ways

The preferred embodiments of the present invention are specifically described below with reference to the accompanying drawings, wherein the accompanying drawings constitute a part of the present invention, and together with the embodiments of the present invention, are used to explain the principles of the present invention, but not to limit the scope of the present invention.

The invention provides a molybdenum-based nanozyme with high catalytic activity, and the preparation raw materials of the high catalytically active molybdenum-based nanozyme include molybdenum oxide nanoparticles, cysteine and copper ions.

Specifically, the raw material for the preparation of the highly catalytically active molybdenum-based nanozyme also includes a surfactant.

Specifically, the above-mentioned surfactant is a polymer with good biocompatibility, for example, polyvinylpyrrolidone (PVP).

The present invention also provides a method for preparing a platinum-based nanozyme with high catalytic activity, comprising the following steps:

Step S1, preparing molybdenum oxide nanoparticles;

Step S2, adding cysteine (L-Cys) and water-soluble copper salt to the 20mL secondary aqueous solution, and ultrasonically stirring and chelating into a white floc L-Cys-Cu; wherein, the water-soluble copper salt is copper chloride ( CuCl ₂ ); the amount of cysteine (L-Cys) is 2 mmol, and the amount of copper chloride (CuCl ₂ ) is 1 mmol;

In step S3, the molybdenum oxide nanoparticles and L-Cys-Cu are mixed and placed in a centrifuge tube, placed in an ultrasonic bath for 3-8 minutes, and then placed on a magnetic stirrer for stirring. During the stirring process, the surface active agent is added slowly and uniformly. agent PVP and self-assemble;

Step S4, after the reaction is completed, the precipitate is taken out by centrifugation, the precipitate is repeatedly centrifugally washed 2-3 times with secondary water, and then centrifuged once with ethanol, and excess ethanol is sucked off to obtain a high catalytic activity platinum-based nanozyme (MoO ₂ - L-Cys-Cu-PVP).

Specifically, in the above step S1, the steps of preparing molybdenum oxide nanoparticles include:

S101, weighing ammonium molybdate powder in proportion, dissolving the ammonium molybdate powder in deionized water, and ultrasonically dissolving to obtain an aqueous solution of ammonium molybdate;

S102, slowly adding absolute ethanol to the ammonium molybdate aqueous solution obtained in S101 using a pipette, ultrasonically dispersing and stirring at room temperature for 30-40min to obtain a uniformly mixed mixed solution;

S103, transfer the mixed solution into the hydrothermal reaction kettle of the polytetrafluoroethylene liner, tighten the reaction kettle and place it in an oven, set the reaction temperature to be 160-200°C, and the reaction time to be 10-15h;

S104. After the reaction is completed, after the reaction is naturally cooled to room temperature, the precipitate is taken out by centrifugation, and the precipitate is repeatedly centrifuged and washed with secondary water for 2-5 times, and then placed in a freeze dryer for lyophilization for 45-50 hours to obtain molybdenum oxide (MoO ₂ ) nanometers. particles.

It should be noted that, in the above S101 and S102, ammonium molybdate: absolute ethanol: deionized water = 0.7-1 mmoL: 10-15 mL: 20-25 mL.

Specifically, in the above S104, the surface of the molybdenum oxide (MoO ₂ ) nanoparticles has a rough porous structure.

Specifically, in the above step S2, the molar ratio of cysteine and copper chloride is controlled to be 2:1-1.5. Preferably, the molar ratio of cysteine and copper chloride is 2:1.

Specifically, in the above step S3, the molar ratio of the molybdenum oxide nanoparticles to the L-Cys-Cu is controlled to be 1:1-3.

Specifically, in the above step S3, the mass ratio of molybdenum oxide nanoparticles to PVP is controlled to be 1:1-2.

Specifically, in the above step S4, the structure of the highly catalytically active molybdenum-based nanozyme is that copper ions are dispersed and adsorbed on the surface of the molybdenum oxide nanoparticles, that is, the porous molybdenum oxide nanoparticles and cysteine (L-Cys)-copper ions are realized. effective anchoring.

On the other hand, the present invention also provides the application of a highly catalytically active molybdenum-based nanozyme as an anti-tumor material.

Compared with the prior art, the present invention utilizes a simple one-step hydrothermal method, decomposes ammonium molybdate under hydrothermal conditions and re-nucleates and grows into porous MoO2 nanoparticles with high catalase _- like catalysis. Active, degrading behavior during catalysis and able to avoid long-term toxicity of nanomaterials to organisms, this is a safe and efficient H2O2 _- responsive _nanoenzyme catalyst in TME, and can improve intratumor hypoxia, sensitize Radiotherapy; in the process of anchoring copper ions on the surface of MoO ₂ nanoparticles, only simple and safe ultrasonic stirring at room temperature, and no other high temperature and high pressure processes to change the morphology and structure of MoO ₂ nanoparticles, through cysteine L -Cys acts as an intermediate linker for ion anchoring, which provides a simple reference for other transition metal oxides to anchor metal ions.

The highly catalytically active molybdenum-based nanozyme (abbreviation: MCCP) of the present invention has synergistically enhanced catalase-like catalytic decomposition of H ₂ O ₂ to generate oxygen to improve the effects of intratumoral hypoxia and enhanced radiotherapy. In the two aqueous solutions (MP, MCCP) with the same mass content of MoO, MCCP catalyzed the decomposition of H ₂ O ₂ in the same time as about twice as much as that of the single MP experimental group without L-Cys-Cu _; The aqueous solution of L-Cys-Cu alone at the experimental concentration of ₂ O ₂ has no obvious catalase-like catalytic effect, which may be due to the larger size of L-Cys-Cu particles and fewer exposed active sites. However, after adsorbing it _on the surface of MoO2 nanoparticles with large surface area, high dispersion of L _- Cys _- Cu was achieved, exposing more copper active sites in contact with H2O2, thus achieving low The effective catalase-like catalytic activity of MCCP at a concentration of MCCP produces a large amount of oxygen, improves intratumoral hypoxia, and achieves radiosensitization.

The catalase catalytic activity and radiosensitization function of the highly catalytically active molybdenum-based nanozyme provided by the present invention are much higher than those of molybdenum oxide alone and L-Cys-Cu alone, and the used dose of H ₂ O ₂ is 100 μM -1.0 mM, in the high expression dose range of H2O2 in _TME , while improving the _{catalase -} like catalytic ability of _MCCP for relatively low concentrations of H2O2, while also reducing the relatively high biotoxicity of copper The dosage of ions and the dosage of H ₂ O ₂ finally obtained molybdenum-based nanozymes with excellent catalase-like mimetic catalytic activity. After MCCP catalyzes H ₂ O ₂ to generate oxygen, which is easily disassembled in TME under X-ray irradiation during radiotherapy, L-Cys-Cu is released from the composite and begins to undergo a Fenton-like reaction, resulting in more Therefore, MCCP improves tumor hypoxia and also improves tumor sensitivity during radiotherapy, so it has broad application prospects in tumor radiosensitization.

It is worth noting that the principle of the application of the highly catalytically active platinum-based nanozyme as an anti-tumor material is: using H ₂ O ₂ highly expressed in the tumor or H ₂ O ₂ provided by exogenous sources (for example, during radiotherapy) The X-ray irradiation process will increase the expression of H ₂ O ₂ in the tumor), and combined with the valence state conversion of both Cu ²⁺ and Cu ¹⁺ (ie: Fenton-like reaction), the decomposition reaction of H ₂ O ₂ can be cyclically catalyzed Produce highly toxic hydroxyl radicals (·OH), ·OH has strong oxidative properties, can kill cancer cells more effectively, and the reaction process can generate O ₂ .

Fenton-like reaction of copper ions:

Cu ²⁺ +H ₂ O ₂ =Cu ¹⁺ +O ₂ +H ₂ O

Cu ²⁺ +H ₂ O ₂ =Cu ¹⁺ +·OH

Example 1

The present embodiment provides a molybdenum oxide nanoparticle, and the preparation method is as follows:

(1) Weigh 0.7mmol (0.0865g) of ammonium molybdate powder and dissolve it in 20mL of deionized water, and ultrasonically dissolve to obtain an aqueous solution of ammonium molybdate;

(2) use a 5mL pipette to slowly add 10mL of dehydrated alcohol to the aqueous solution of ammonium molybdate in step (1), ultrasonically disperse and stir at room temperature for 30 minutes to obtain a fully mixed mixed solution; wherein, dehydrated alcohol and The volume ratio of water is 1:2, and the total volume of the mixed solution is 30 mL;

(3) the above-mentioned mixed solution of 30mL is transferred into the hydrothermal reaction kettle of the polytetrafluoroethylene liner of 50mL, after the reaction kettle is tightened, it is placed in an oven, and the setting reaction temperature is 160 ℃, and the reaction time is 12h;

(4) After the reaction, after the reaction was naturally cooled to room temperature, the precipitate was taken out by centrifugation, washed with secondary water for three times by centrifugation, and lyophilized in a freeze dryer for 48 hours to obtain molybdenum oxide nanoparticles (MoO ₂ ).

Example 2

The present embodiment provides a high catalytic activity platinum-based nanozyme, and the preparation method is as follows:

(1) MoO synthesized in Example ₁ and L-Cys-Cu were mixed in a 20 mL aqueous solution with a molar ratio of 1:1, ultrasonicated for 3 minutes in a centrifuge tube, and then placed on a magnetic stirrer to stir;

(2) During the stirring process, add the surfactant PVP evenly and slowly (the mass ratio of MoO to PVP is ₁ :1);

(3) after the reaction is completed, centrifuge out the precipitate, repeat centrifugal washing with secondary water for 2-3 times, wash with ethanol and centrifuge, and absorb excess ethanol to obtain a high catalytic activity platinum-based nanozyme (MoO ₂ -L- Cys-Cu-PVP, referred to as MCCP).

It should be noted that, step (3) includes placing the MoO ₂ at the bottom of the obtained centrifuge tube in a 4° C. refrigerator for later use.

Comparative Example 1

This comparative example provides a MoO ₂ -PVP, and the preparation method is as follows:

At room temperature, 100-300 mg of MoO ₂ powder synthesized in Example 1 was added to 20 mL of secondary aqueous solution, and the surfactant PVP was evenly and slowly added during stirring to obtain MoO ₂ -PVP (abbreviation: MP). Among them, the mass ratio of MoO ₂ to PVP is 1:1.

Test Example 1

Scanning electron microscopy and transmission electron microscopy observations.

As shown in Figure 1: a and b are the scanning electron microscope and transmission electron microscope images of the MCCP nanoparticles of the present invention, respectively. It can be seen that the size of the nanoparticles is about 150 nm, and each nanoparticle is composed of particles of smaller size. There is a certain gap between the small particles, which shows an obvious porous structure; c is the element energy spectrum (EDX) of MCCP, indicating that the main elements of MCCP are Mo and Cu; d is the X-ray powder diffraction of MCCP ( XRD) pattern, its peak position can correspond to the standard card of MoO ₂ (JCPDS: 50-0739).

Test case 2

Evaluation of catalase-like catalytic activities of MoO ₂ , L-Cys-Cu and MCCP.

First, ultrasonically dissolve MP of different masses (final concentrations of 2.5, 5, 10, and 20 μg/mL) in deionized water, and immerse the reset probe of the dissolved oxygen tester in the MP water solution. After the instrument reading is stable, Record the reading of the instrument at this time, that is, the original oxygen content in the aqueous solution, that is, the initial zero time point in the process of catalytic oxygen production, and then add hydrogen peroxide with a final concentration of 1.0mM. After the catalytic process starts, carry out every 15 seconds. data record. As shown in a in Figure 2, the dissolved oxygen test results of MP nanoparticles with different concentrations show that the catalase-like catalytic activity of MP nanoparticles is positively correlated with the concentration of MP; Compared with MP, the MCCP has an enhanced catalase-like catalytic effect. The following comparative experiments were carried out: set up three experimental groups, namely MP, MCCP and L-Cys-Cu. The specific process is as follows: the same volume of 50 μg/mL _The MoO2 aqueous solution and the 400 μg/mL L-Cys-Cu aqueous solution were divided into two equal volumes and placed in a centrifuge tube, and then one part was taken from each of the two solutions and stirred to synthesize MCCP nanoparticles (MCCP nanoparticle composition). MoO2 concentration in 25 μg/mL and L-Cys-Cu into ₂₀₀ μg/mL), then the remaining part of _MoO2 aqueous solution was synthesized as MP nanoparticles, and deionized water was added to the MoO2 concentration of ₂₅ μg/mL , the remaining 400 μg/mL L-Cys-Cu aqueous solution only needs to add an equal volume of deionized water to reduce the concentration to 200 μg/mL. At this time, the three groups of solutions are MCCP (MoO ₂ 25 μg/mL and L-Cys -Cu 200 μg/mL), MP (MoO ₂ 25 μg/mL), L-Cys-Cu aqueous solution (200 μg/mL), and then, according to the previous steps, the oxygen production measurement of the dissolved oxygen tester was performed. As shown in Figure 2, b is the consumption curve of hydrogen peroxide by MCCP using titanium sulfate as the indicator, wherein the inset is the colorimetric optical photo of the color change of the indicator during the consumption of H ₂ O ₂ by MCCP under the instruction of titanium sulfate. (The titanium sulfate solution itself is colorless, and the H2O2 reacts with titanium _sulfate to become yellow (UV _- visible absorption peak is _407nm ), as the H2O2 is consumed by MCCP, the titanium _sulfate remains colorless), the color of the inset varies with It changes from yellow to colorless with time, which indicates that H ₂ O ₂ is continuously consumed with time; in Fig. 2, c is the catalase-like enzyme of three materials MP, MCCP, L-Cys-Cu The comparison of the catalytic activity of catalyzing the decomposition of H ₂ O ₂ ; the results show that the MCCP obtained by the composite of L-Cys-Cu and MoO ₂ can significantly enhance the catalase-like activity of the two, that is, the ability to catalyze the decomposition of H ₂ O ₂ ; In Figure 2, d is the effect of temperature on the catalytic effect of MCCP; it shows that the reaction is an endothermic reaction, and the reaction rate increases with the increase of temperature.

Test case 3

Intracellular reactive oxygen species (ROS) content assessment.

The ROS probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA, 10 μM, Beyotime Co, Sigma-Aldrich, USA) was used to evaluate the catalytic activity of MCCP nanoparticles under X-ray irradiation. Acetylated and oxidized to 2′,7′-dichlorofluorescein (DCF) with high fluorescence properties. First, adenocarcinoma human alveolar basal epithelial cells (A549 cells) were adherently cultured at a cell concentration of 2×10 ⁵ /dish for 24 hours in complete medium, and then two groups of cells were added with MCCP: 50 μg/mL, respectively. MP: 50 μg/mL, no material was added to another group of cells, only the same volume of complete medium was added to serve as a control group, 5 hours later, washed twice with PBS, and 200 μL of probe solution (1 μL DCFH-DA) was added to each small dish +999μL of RPMI cell culture medium) was incubated in the incubator for 20 minutes, then washed with PBS, and the experimental group was irradiated by X-ray (50kv, 49μA) for 15 minutes, and then the nucleus was stained with Hoechst33342 (Beyotime)15 minute. Finally, they were washed twice with PBS and observed under a laser confocal microscope (A1/LSM-Kit, Nikon/Pico Quant GmbH, Japan/Germany).

Fig. 3 is a Confocal Laser Scanning Microscopy (CLSM) image of the detection of reactive oxygen species (ROS) at the cellular level by MP and MCCP, indicating that MCCP has the strongest free radicals under the excitation of X-ray. This may be due to the synergistic catalysis of copper ions and molybdenum oxide to increase the level of _O2 in hydrogen peroxide production, which improves tumor hypoxia, which in turn promotes ROS production. Another reason is that copper ions on L-Cys-Cu are After the gradually degraded molybdenum oxide surface is released, photo-excited Fenton-like reaction occurs under the activation of X-ray to generate more OH, thereby sensitizing the radiotherapy of cancer cells.

Test Example 4

Evaluation of the killing of cancer cells by an in vitro DNA double-strand break assay.

First, A549 cells were seeded in a 24-well plate with a cell concentration of 3×10 ⁴ /well. After 24 hours of incubation, the cells were divided into three groups, including: MCCP was added to the two groups of cells: 50 μg/mL. , MP: 50μg/mL, another group of cells was not added with material, only the same volume of complete medium was added as a control group, and 5 hours later, X-ray (50kv, 49μA) irradiation was performed for 15 minutes. Cells were washed twice with PBS and fixed with 4% paraformaldehyde, then washed three times with PBS for 3 minutes on a shaker, and pretreated by adding 0.2% Triton-X-100 (300 μL, 10 minutes) solution To increase the permeability of the membrane, dilute the primary antibody (β-Actin, backbone protein antibody) with blocking solution (5% FBS, 1% Triton-X-100), the dilution ratio is 1:100, and configure it as a primary antibody diluent, After the dilution, the blocking solution in the wells was discarded, and 200 μL of primary antibody diluent was added to each well. Finally, the 24-well plate was wrapped in gauze soaked in PBS and kept in a refrigerator at 4°C overnight. The 24-well plate was removed from the refrigerator and washed three times with PBS in a shaker for 3 min each. Subsequently, secondary antibody dilution (anti-rabbitAlexaFluor-488conjugatedIgG:PBS=1:500, 200 μL per well, temperature 37°C, duration 1 hour) was added, and the nuclei were stained with Hoechst after washing three times with PBS, and finally washed with PBS. After three times, coverslips were used to cover the slides and placed under a laser confocal microscope to observe, image and record data.

Figure 4 is a confocal laser scanning micrograph of an in vitro DNA double-strand break assay. The results show that, consistent with the in vitro ROS analysis, MCCP nanoparticles have stronger DNA damage ability, that is, stronger anti-tumor ability.

Test Example 5

_In vitro detection of MCCP _- catalyzed H2O2 production of hydroxyl radicals.

Design considerations: Considering that not only H ₂ O ₂ is highly expressed in tumors, but also GSH, as an antioxidant, is also expressed in tumors. Therefore, this experiment simulates the highly expressed GSH in the tumor microenvironment, and GSH can act as a reducing agent , the Cu ²⁺ in MCCP is reduced to Cu ¹⁺ , and Cu ¹⁺ has stronger Fenton-like reaction ability under the action of X-ray, so that H ₂ O ₂ can be decomposed into hydroxyl radicals. First, prepare carbonate buffer solution (BBS) with a concentration of 0.1 mol/L, and then use BBS solution to prepare DTNB (a probe for detecting-SH) solution with a concentration of 100 mM, 1.0 mM glutathione (GSH) solution ) solution, and MP and MCCP solutions of different concentrations, and then prepare 100 mM Tris HCL solution with secondary water. After all solutions are prepared, Ellman's analysis is performed. First, 225 μL of GSH solution and 225 μL of MCCP/MP solution are mixed. Mix, shake for 3 hours in the dark, and then add 785 μL of Tris diluent and 15 μL of DTNB solution, each with a volume of 1250 μL, and measure the absorbance at 412 nm. The reaction mechanism is shown in Figure 7.

The OH produced by MCCP under the action of X-ray is detected by OH probe (terephthalic acid, TA, 5mM, AlfaAesar), TA can react with OH to generate 2-hydroxyterephthalic acid (TAOH, maximum fluorescence peak at 435 nm). First, the prepared TA solution was added to the divided groups with/without H ₂ O ₂ (Control, MP, MCCP), and the hydrogen peroxide was 1.0 mM, in which the MCCP solution was first reacted with excess GSH to ensure that all the Cu ²⁺ Converted to Cu ¹⁺ , and then placed in a shaker at 37°C overnight, the change in the fluorescence emission front was recorded at 435 nm.

As shown in Figure 5: a is the consumption curve of GSH with different concentrations of MCCP nanoparticles at the same time, indicating that the consumption rate of GSH is positively correlated with the concentration of MCCP nanoparticles; b is the control experiment of MP, PBS and MCCP, indicating that only The MCCP experimental group can only consume GSH, that is, the presence of Cu ²⁺ causes redox reaction with GSH; c is the consumption curve of GSH with time under the same MCCP nanoparticle concentration, indicating that the consumption of GSH is positively correlated with time; d is X- Under ray irradiation, the fluorescence emission spectrum of the product·OH in the Fenton-like reaction of Cu ²⁺ and H ₂ O ₂ was significantly higher in the TA+H ₂ O ₂ +MCCP group than in the TA+H ₂ O ₂ +MP group. The fluorescence intensity shows that under X-ray irradiation, the Fenton-like reaction of copper ions in MCCP produces ·OH, but the reason for lower than TA+H ₂ O ₂ may be that part of H ₂ O ₂ is catalyzed to generate O ₂ . This results in a reduction in the total amount of fixed H ₂ O ₂ , which in turn produces relatively less ·OH.

As shown in Figure 6, Figure 6 shows the cytotoxicity data of MCCP nanoparticles and human umbilical vein epithelial cells HUVEC co-incubated for 24 hours. The test results show that MCCP has no obvious cytotoxicity within the tested dose range.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. Substitutions should be covered within the protection scope of the present invention.

Claims (8)

1. The molybdenum-based nanoenzyme with high catalytic activity is characterized by comprising molybdenum oxide nanoparticles and copper ions, wherein the copper ions are anchored on the surfaces of the molybdenum oxide nanoparticles;

the preparation method of the molybdenum-based nanoenzyme with high catalytic activity comprises the following steps:

step S1, preparing molybdenum oxide nano particles;

step S2, adding cysteine and water-soluble copper salt into secondary water, ultrasonically stirring and chelating to synthesize white floccule L-Cys-Cu;

s3, placing the molybdenum oxide nano-particles and the L-Cys-Cu mixed solution into a centrifuge tube, carrying out ultrasonic treatment in an ultrasonic pool, then placing the centrifuge tube on a magnetic stirrer for stirring, and uniformly and slowly adding a surfactant in the stirring process;

and step S4, after the reaction is finished, centrifuging to take out the precipitate, repeatedly centrifuging and washing the precipitate for 2-3 times by using secondary water, washing by using ethanol and centrifuging, and sucking off redundant ethanol to obtain the molybdenum-based nanoenzyme with high catalytic activity.

2. The molybdenum-based nanoenzyme with high catalytic activity as claimed in claim 1, wherein the surfactant is a polymer with good biocompatibility.

3. The method for preparing molybdenum-based nanoenzyme with high catalytic activity as claimed in claim 1, comprising the steps of:

step S1, preparing molybdenum oxide nano particles;

step S2, adding cysteine and water-soluble copper salt into secondary water, ultrasonically stirring and chelating to synthesize white floccule L-Cys-Cu;

s3, putting the mixed solution of the molybdenum oxide nano-particles and L-Cys-Cu into a centrifugal tube, carrying out ultrasonic treatment in an ultrasonic pool, then putting the ultrasonic pool on a magnetic stirrer for stirring, and uniformly and slowly adding a surfactant in the stirring process;

and step S4, after the reaction is finished, centrifuging and taking out the precipitate, repeatedly centrifuging and washing the precipitate for 2-3 times by using secondary water, washing by using ethanol and centrifuging, and sucking away excessive ethanol to obtain the molybdenum-based nanoenzyme with high catalytic activity.

4. The method according to claim 3, wherein the step S1 of preparing the molybdenum oxide nanoparticles comprises:

s101, weighing ammonium molybdate powder according to a proportion, dissolving the ammonium molybdate powder in deionized water, and performing ultrasonic dissolution to obtain an ammonium molybdate aqueous solution;

s102, adding absolute ethyl alcohol into the ammonium molybdate aqueous solution obtained in the S101 by using a liquid-transferring gun, performing ultrasonic dispersion, and stirring at room temperature to obtain a uniformly mixed solution;

s103, transferring the mixed solution into a hydrothermal reaction kettle, screwing the reaction kettle, placing the reaction kettle in an oven, setting the reaction temperature to 160-200 ℃, and the reaction time to 10-15 h;

and S104, after the reaction is finished, naturally cooling to room temperature, centrifuging, taking out the precipitate, repeatedly centrifuging and washing the precipitate for 2-5 times by using secondary water, and freeze-drying in a freeze dryer to obtain the molybdenum oxide nanoparticles.

5. The method according to claim 4, wherein in S101 and S102, the ratio of ammonium molybdate: anhydrous ethanol: deionized water =0.7-1 mmoL: 10-15 mL: 20-25 mL.

6. The method according to claim 3, wherein in step S2, the water-soluble copper salt is cupric chloride, and the molar ratio of cysteine to cupric chloride is 2: 1-1.5.

7. The method according to claim 3, wherein in the step S3, the molar ratio of the molybdenum oxide nanoparticles to L-Cys-Cu is 1: 1-3.

8. The method according to any one of claims 3 to 7, wherein in the step S3, the surfactant is polyvinylpyrrolidone, and the mass ratio of the molybdenum oxide nanoparticles to the polyvinylpyrrolidone is 1: 1-2.

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