Preparation method of biodegradable mn-zno nanoclusters with porous structure and enzyme-like activity

Abstract

The application relates to a preparation method of Mn-ZnO, and relates to a preparation method of biodegradable Mn-ZnO nanoclusters with a porous structure and enzyme-like activity. The application aims at solving the problem of low piezoelectric catalytic efficiency of the existing ZnO nanomaterials. The method comprises the following steps: firstly, stirring and mixing Zn(NO3)2.6H2O, Mn(NO3)2 and diethylene glycol; secondly, heating and refluxing; thirdly, centrifuging, washing, drying and grinding. The application is used for the preparation of biodegradable Mn-ZnO nanoclusters with a porous structure and enzyme-like activity.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing Mn-ZnO. BACKGROUND

[0002] Sonodynamic therapy (SDT) is a new treatment mode that sonosensitizer generates reactive oxygen species (ROS) under the irradiation of ultrasound (US) to cause tumor cell apoptosis. SDT has attracted more and more attention due to its high tissue penetration depth and non-invasive advantages. Undoubtedly, sonosensitizer plays a crucial role in SDT. Compared with organic sonosensitizers, inorganic sonosensitizers have the advantages of low cost, strong stability, and unique imaging ability and biological activity. Moreover, some inorganic sonosensitizers can generate hydroxyl radicals by Fenton or Fenton-like reaction with excess hydrogen peroxide in the tumor microenvironment, causing tumor cell apoptosis. However, the inherent toxicity of inorganic nanomaterials and the long-term biological toxicity risk caused by their accumulation in vivo are the biggest obstacles for inorganic sonosensitizers to move towards clinical translation. At present, the sonosensitizers reported in the literature mainly include titanium dioxide-based and bismuth-based materials, and most of these materials are non-degradable, which greatly limits their clinical application. Therefore, it is of great research significance and practical value to develop biodegradable inorganic sonosensitizers. In addition, the current sonodynamic therapy still has the problem of low efficiency of reactive oxygen species. The main reason is that the efficiency of electron-hole separation generated by sonosensitizer under ultrasonic irradiation is low. In addition, as an oxygen-dependent treatment mode, the treatment effect of sonodynamic therapy is seriously dependent on the concentration of oxygen. However, the tumor microenvironment is a hypoxic environment, which seriously restricts the effect of sonodynamic therapy.

[0003] As a non-centrosymmetric crystal, piezoelectric material is becoming a new type of sonosensitizer. Under the action of external mechanical strain, piezoelectric material produces built-in electric field due to the separation of positive and negative charge centers on the two opposite surfaces, and in the absence of external strain, it shows zero electric dipole. Therefore, the built-in electric field can promote the separation of carriers and inhibit the recombination of carriers, and the generated excited state electrons and holes immediately react with H2O or O2 to generate various types of active oxygen (such as ·OH and ·O2-). Unlike traditional sonosensitizers that require the consumption of O2 to generate active oxygen, piezoelectric materials mainly react with H2O or O2 through an electron transfer pathway to generate ROS, which has the characteristics of not or less dependent on O2 concentration, and can overcome the limitation of hypoxia in tumor microenvironment. In recent years, several traditional piezoelectric materials such as BaTiO3, MoWOx, ZnO and 2D bismuth-based materials have been reported for tumor treatment. However, most of the materials cannot be degraded in vivo, which can easily lead to long-term biological toxicity. As a typical piezoelectric semiconductor material, ZnO has the advantages of good biocompatibility and degradability under acidic conditions, and has broad application prospects in the field of biomedicine. However, its piezoelectric catalytic performance still needs to be further improved. Introducing defects or vacancies through ion doping is one of the effective means to improve the piezoelectric catalytic efficiency of piezoelectric materials. However, most of the metal ion doped ZnO nanoparticles reported in the literature are first prepared by the precipitation method to generate zinc oxide precursors, and then high-temperature calcination in a muffle furnace. This method not only has complicated steps, but also easily leads to the lack of surface groups of ZnO during high-temperature calcination, which is not conducive to biological applications. In addition, the ZnO nano-materials reported at present are mainly rod-like structures, and there is no report on ion-doped ZnO with porous structure, while porous structure has more catalytically active sites and faster carrier migration speed, which is conducive to improving the catalytic efficiency of the material. SUMMARY

[0004] The present application aims to solve the problem of low piezoelectric catalytic efficiency of existing ZnO nano-materials, and further provides a preparation method of biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity.

[0005] The preparation method of biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity is carried out according to the following steps:

[0006] I. Under the condition of temperature 40℃-60℃, Zn(NO3)2·6H2O, Mn(NO3)2 and diethylene glycol are stirred and mixed uniformly to obtain a mixed solution;

[0007] II. Under stirring, the mixed solution is heated to 210℃-230℃ at a heating rate of 1℃ / min-2℃ / min, and heated to reflux for 10h-14h under the condition of temperature 210℃-230℃, and cooled to room temperature to obtain a brown suspension.

[0008] Three, add anhydrous ethanol to the brown suspension, then centrifuge to remove the supernatant, obtain the precipitate, and then wash, dry and grind the precipitate, to complete the preparation method of the biodegradable Mn-ZnO nanocluster with porous structure and enzyme-like activity.

[0009] The beneficial effects of the present application are:

[0010] The present application adopts one-pot method to synthesize biodegradable porous Mn-ZnO nanocluster with multiple enzyme activities (POD and OXD activities), and is used for enzyme catalysis and piezoelectric catalysis tumor treatment, which has the following advantages:

[0011] One, compared with most piezoelectric materials, the nanocluster has good biocompatibility and degradability (weak acid), can avoid long-term accumulation of metal ions in the body, and has good clinical transformation prospect.

[0012] Two, unlike high-temperature calcination method, the present application adopts thermal decomposition of zinc nitrate and manganese nitrate in high-boiling solvent diethylene glycol (DEG), which endows the ZnO surface with more hydrophilic groups (such as hydroxyl groups, etc.), which is beneficial to biological application. On the contrary, high-temperature calcination method easily leads to loss of functional groups on the surface of ZnO, resulting in serious decrease of hydrophilicity.

[0013] Three, the synthesis method of the nanocluster is simple, does not need multi-step synthesis, has mild reaction conditions, is easy to operate, and has high yield (up to gram level), which is suitable for large-scale production.

[0014] Four, the nanocluster not only has good piezoelectric properties, but also has peroxidase (POD) and oxidase (OXD) activities, which can form active oxygen storm in tumor cells and cause tumor cell apoptosis. In addition, the porous structure, good hydrophilicity and non-toxicity of the material can effectively load various drug molecules.

[0015] Five, the nanocluster can also consume excess glutathione (GSH) expressed in the tumor microenvironment, which can avoid the consumption of GSH to generate active oxygen.

[0016] Six, the Mn-ZnO prepared by the method has a large number of oxygen vacancy defects on the surface, which inhibits the recombination of electrons and holes under ultrasonic action, and improves the piezoelectric catalytic performance of the material. In addition, the existence of oxygen vacancies is beneficial to the adsorption and desorption of H2O2 molecules, and is beneficial to improve the POD enzyme catalytic activity.

[0017] Due to the piezoelectric property of the porous nanocluster, a large amount of active oxygen can be generated under the action of ultrasound, and is less affected by the hypoxic tumor microenvironment. In addition, the material can also react with the excess hydrogen peroxide in the tumor microenvironment to generate hydroxyl radicals (·OH) with strong biological toxicity, and then form an active oxygen storm in the tumor cells, so it can be used for efficient tumor treatment.

[0018] The application relates to a preparation method of a biodegradable Mn-ZnO nanocluster with a porous structure and enzyme-like activity. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 TEM photos of the Mn-ZnO nanoclusters prepared in Example 1;

[0020] Figure 2 STEM mapping photos of the Mn-ZnO nanoclusters prepared in Example 1;

[0021] Figure 3 High-resolution TEM photos of the Mn-ZnO nanoclusters prepared in Example 1;

[0022] Figure 4 XRD spectra of the Mn-ZnO nanoclusters prepared in Example 1 and undoped ZnO prepared in a comparative experiment;

[0023] Figure 5 High-resolution XPS spectra, a is a Mn2p spectrum of the Mn-ZnO nanoclusters prepared in Example 1, b is an O2s spectrum of undoped ZnO prepared in a comparative experiment and the Mn-ZnO nanoclusters prepared in Example 1;

[0024] Figure 6 Electron paramagnetic resonance spectra of undoped ZnO prepared in a comparative experiment and the Mn-ZnO nanoclusters prepared in Example 1;

[0025] Figure 7 Low-temperature nitrogen adsorption-desorption isotherms and adsorption pore size distribution diagrams of the Mn-ZnO nanoclusters prepared in Example 1, a is a low-temperature nitrogen adsorption-desorption isotherm, and b is an adsorption pore size distribution diagram;

[0026] Figure 8 Piezoelectric phase diagrams of undoped ZnO prepared in a comparative experiment and the Mn-ZnO nanoclusters prepared in Example 1;

[0027] Figure 9 Acoustic current curve diagrams of ZnO with different Mn doping amounts, 1 is undoped ZnO prepared in a comparative experiment, 2 is 1%-Mn-ZnO, 3 is 4%-Mn-ZnO, and 4 is 2%-Mn-ZnO;

[0028] Figure 10 The values ​​represent the electrochemical impedance of ZnO with different Mn doping amounts. 1 represents undoped ZnO prepared in the comparative experiment, 2 represents 1%-Mn-ZnO, 3 represents 2%-Mn-ZnO, and 4 represents 4%-Mn-ZnO.

[0029] Figure 11 The piezoelectric catalytic performance evaluation diagram of undoped ZnO and 2%-Mn-ZnO prepared by comparison experiment using DPBF as a probe is shown.

[0030] Figure 12 The images show the oxidase activity and electron paramagnetic resonance spectra of 2%-Mn-ZnO under different pH conditions using TMB as a probe. a represents oxidase activity, and b represents electron paramagnetic resonance spectra.

[0031] Figure 13 The images show the peroxidase activity and electron paramagnetic resonance spectrum of 2% Mn-ZnO using TMB as a probe. a represents the peroxidase activity, and b represents the electron paramagnetic resonance spectrum.

[0032] Figure 14 The performance of 2% Mn-ZnO on glutathione (GSH) consumption;

[0033] Figure 15 To evaluate the killing effect of 2%-Mn-ZnO on 4T1 cells using the MTT assay, bar charts were prepared, with 1 representing the blank control group, 2 representing the ultrasound-only group, 3 representing 2%-Mn-ZnO, and 4 representing 2%-Mn-ZnO plus ultrasound.

[0034] Figure 16 Laser confocal micrograph of 2% Mn-ZnO incubated with 4T1 cells;

[0035] Figure 17 Flow cytometry image of 4T1 cells;

[0036] Figure 18 The images show the changes in tumor volume and photographs of the tumors in mice after treatment. a represents the changes in tumor volume, and b represents a photograph of the tumor.

[0037] Figure 19 Blood routine thermograms of healthy mice after injection of 2% Mn-ZnO solution (1 mg / mL);

[0038] Figure 20 Degradation of 2% Mn-ZnO under different acidic conditions;

[0039] Figure 21 The zinc ion content in mouse feces and urine;

[0040] Figure 22 The infrared spectrum of 2% Mn-ZnO. Detailed Implementation

[0041] The technical solution of the present application is not limited to the specific embodiments listed below, but also includes any combination between the specific embodiments.

[0042] Specific embodiment one: the preparation method of the biodegradable Mn-ZnO nanocluster with porous structure and enzyme-like activity in the present embodiment is carried out according to the following steps:

[0043] I. Under the condition of temperature of 40℃-60℃, Zn(NO3)2·6H2O, Mn(NO3)2 and diethylene glycol are stirred and mixed uniformly to obtain a mixed solution;

[0044] II. Under the stirring condition, the mixed solution is heated to 210℃-230℃ at a heating rate of 1℃ / min-2℃ / min, and under the condition of temperature of 210℃-230℃, it is heated to reflux for 10h-14h, and then cooled to room temperature to obtain a brownish suspension;

[0045] III. Anhydrous ethanol is added to the brownish suspension, and then the supernatant is removed by centrifugation to obtain a precipitate, which is washed, dried and ground in sequence, thereby completing the preparation method of the biodegradable Mn-ZnO nanocluster with porous structure and enzyme-like activity.

[0046] In the present embodiment, mesoporous Mn-doped ZnO nanoclusters with a large number of oxygen vacancies are prepared for the first time by a simple doping strategy and a thermal decomposition (220℃) of metal salt precursors in a high-boiling-point solvent diethylene glycol (DEG). The unique porous structure of the Mn-ZnO nanoclusters in the present embodiment not only increases the catalytic active sites, but also promotes the separation of electrons and holes, thereby being conducive to the pressure catalytic performance and enzyme-like catalytic activity of Mn-ZnO. First, after Mn doping, the lattice of Mn-ZnO is distorted, thereby causing polarization enhancement and improving the piezoelectric performance. In addition, the oxygen vacancies generated due to Mn doping promote the separation of ultrasound-induced electron-hole pairs. Therefore, Mn-doped ZnO can effectively generate reactive oxygen species under ultrasonic irradiation. Second, the doped Mn ions exhibit excellent TME-responsive multi-enzyme mimic (POD and OXD-like) activity, which further generates reactive oxygen species in the tumor microenvironment. Third, Mn-doped ZnO exhibits significant GSH consumption ability due to the existence of mixed valence of Mn 2+ / Mn 3+ , which avoids reducing the level of intracellular reactive oxygen species.

[0047] The beneficial effects of the present embodiment are:

[0048] The present embodiment adopts one-pot method to synthesize the multi-enzyme activity (POD and OXD activity) biodegradable porous Mn-ZnO nanoclusters, and is used for enzyme catalysis and piezoelectric catalysis tumor treatment, which has the following advantages:

[0049] I. Compared with most piezoelectric materials, the nanoclusters have good biocompatibility and degradability (weak acid), can avoid long-term accumulation of metal ions in the body, and have good clinical transformation prospects.

[0050] II. Unlike high-temperature calcination method, the present method adopts thermal decomposition of precursor zinc nitrate and manganese nitrate in high-boiling solvent diethylene glycol (DEG), which endows the ZnO surface with more hydrophilic groups (such as hydroxyl groups, etc.), which is beneficial to biological application. On the contrary, high-temperature calcination method easily leads to loss of functional groups on the surface of ZnO, resulting in a serious decrease in hydrophilicity.

[0051] III. The synthesis method of the nanoclusters is simple, does not need multi-step synthesis, has mild reaction conditions, is easy to operate, and has high yield (up to gram level), which is suitable for large-scale production.

[0052] IV. The nanoclusters not only have good piezoelectric properties, but also have peroxidase (POD) and oxidase (OXD) activities, which can form active oxygen storm in tumor cells and cause tumor cell apoptosis. In addition, the porous structure, good hydrophilicity and non-toxicity of the material can effectively load various drug molecules.

[0053] V. The nanoclusters can also consume excess glutathione (GSH) expressed in the tumor microenvironment, which can avoid the consumption of GSH to generate active oxygen.

[0054] VI. The Mn-ZnO prepared by the method has a large number of oxygen vacancy defects on the surface, which inhibits the recombination of electrons and holes under ultrasonic action, and improves the piezoelectric catalytic performance of the material. In addition, the existence of oxygen vacancies is beneficial to the adsorption and desorption of H2O2 molecules, which is beneficial to improve the POD enzyme catalytic activity.

[0055] Because the porous nanoclusters have piezoelectric properties, under the action of ultrasound, they can generate a large amount of active oxygen and are less affected by the hypoxic tumor microenvironment. In addition, the material can also react with the excess hydrogen peroxide in the tumor microenvironment to generate hydroxyl radicals (·OH) with strong biological toxicity, and then form an active oxygen storm in the tumor cells, so it can be used for efficient tumor treatment.

[0056] DETAILED DESCRIPTION TWO: The difference between the present embodiment and the first embodiment is that the mass ratio of Zn(NO3)2·6H2O to Mn(NO3)2 in step one is 1:(0.02-0.12). The others are the same as the first embodiment.

[0057] Embodiment three: different from any one of the embodiment one or two, the mass ratio of Zn(NO3)2.6H2O to Mn(NO3)2 in step one is 1:(0.05-0.12). The others are the same as the embodiment one or two.

[0058] Embodiment four: different from any one of the embodiment one to three, the mass of Zn(NO3)2.6H2O to the volume of diethylene glycol in step one is 1g:(60-80)mL. The others are the same as the embodiment one to three.

[0059] Embodiment five: different from any one of the embodiment one to four, the Zn(NO3)2.6H2O, Mn(NO3)2 and diethylene glycol are stirred for 20min-40min at the temperature of 40℃-60℃ and the stirring speed of 300rpm / min-500rpm / min. The others are the same as the embodiment one to four.

[0060] Embodiment six: different from any one of the embodiment one to five, the mixed solution is heated to 210℃-230℃ at the heating rate of 1℃ / min-2℃ / min at the stirring speed of 300rpm / min-500rpm / min, and heated to reflux for 10h-14h at the temperature of 210℃-230℃. The others are the same as the embodiment one to five.

[0061] Embodiment seven: different from any one of the embodiment one to six, the volume ratio of the brown suspension to anhydrous ethanol in step three is 1:(1-1.5). The others are the same as the embodiment one to six.

[0062] Embodiment eight: different from any one of the embodiment one to seven, the centrifugation to remove the supernatant in step three is specifically centrifuged for 8min-12min at the speed of 80000r / min-10000r / min. The others are the same as the embodiment one to seven.

[0063] Embodiment nine: different from any one of the embodiment one to eight, the washing in step three is specifically washed with anhydrous ethanol for 2-3 times, and then washed with deionized water for 2-3 times. The others are the same as the embodiment one to eight.

[0064] Embodiment ten: different from any one of the embodiment one to nine, the drying in step three is specifically dried for 10h-14h at the temperature of 40℃-50℃ under vacuum. The others are the same as the embodiment one to nine.

[0065] The beneficial effects of the present application are verified by the following examples:

[0066] Example 1

[0067] A method for preparing a biodegradable Mn-ZnO nanocluster with porous structure and enzyme-like activity, which is carried out according to the following steps:

[0068] I. Under the conditions of a temperature of 60℃ and a stirring speed of 300 rpm / min, 1.8 g of Zn(NO3)2·6H2O, 100 mg of Mn(NO3)2 and 108 mL of diethylene glycol are stirred for 30 min to obtain a mixed solution;

[0069] II. Under the condition of a stirring speed of 300 rpm / min, the mixed solution is heated to 220℃ at a heating rate of 2℃ / min, and then heated to reflux at 220℃ for 10 h, and cooled to room temperature to obtain a brownish suspension;

[0070] III. 100 mL of anhydrous ethanol is added to the 100 mL of brownish suspension, and then the supernatant is removed by centrifugation to obtain a precipitate, which is washed, dried and ground in sequence to obtain a Mn-ZnO nanocluster, and the mass percentage of Mn in the Mn-ZnO nanocluster is 2% as measured by ICP, and thus named as 2%-Mn-ZnO.

[0071] The centrifugation to remove the supernatant in step III is specifically carried out at a speed of 10000 rpm / min for 10 min.

[0072] The washing in step III is specifically carried out twice with anhydrous ethanol and then twice with deionized water.

[0073] The drying in step III is specifically carried out at a temperature of 40℃ under vacuum for 12 h.

[0074] It is measured that the yield of the Mn-ZnO nanocluster prepared in Example 1 is 1.5 g.

[0075] Example 2: The difference between this example and Example 1 is that 50 mg of Mn(NO3)2 is added in step I, and a Mn-ZnO nanocluster is obtained in step III, and the mass percentage of Mn in the Mn-ZnO nanocluster is 1% as measured by ICP, and thus named as 1%-Mn-ZnO. The others are the same as in Example 1.

[0076] Example 3: The difference between this example and Example 1 is that 200 mg of Mn(NO3)2was added in Step 1. Step 3 resulted in Mn-ZnO nanoclusters. The mass percentage of Mn in the Mn-ZnO nanoclusters was 4% as measured by ICP. Therefore, it was named 4%-Mn-ZnO. The rest was the same as Example 1.

[0077] Comparative Experiment: The difference between this comparative experiment and Example 1 is that Mn(NO3)2was not added in Step 1. Step 3 resulted in undoped ZnO. The rest was the same as Example 1.

[0078] Figure 1 The TEM image of the Mn-ZnO nanoclusters prepared in Example 1. As can be seen from the figure, the Mn-ZnO has a uniform size, about 140 nm. And the Mn-ZnO nanoclusters are aggregates assembled by smaller nanoparticles (about 7 nm), and there are certain gaps between the particles, thereby giving it a porous structure.

[0079] Figure 2 The STEM mapping image of the Mn-ZnO nanoclusters prepared in Example 1. As can be seen from the figure, the surface of the Mn-ZnO nanoparticles is relatively rough, and it can be clearly seen that Zn, O and Mn elements are uniformly distributed in the Mn-ZnO matrix, confirming that the Mn element is doped into the ZnO.

[0080] Figure 3 The high-resolution TEM image of the Mn-ZnO nanoclusters prepared in Example 1. As can be seen from the figure, the Mn-ZnO has a higher crystallinity, and the crystal lattice fringes are clearly visible. Among them, the interplanar spacing d = 0.26 nm corresponds to the (002) crystal plane of hexagonal zinc oxide. In addition, it can be seen from the figure that Figure 3 b that there are defects (yellow circles) in the ZnO lattice, which are caused by Mn doping.

[0081] Figure 4 The XRD spectrum of the Mn-ZnO nanoclusters prepared in Example 1 and the undoped ZnO prepared in the comparative experiment. It can be seen that the XRD diffraction peaks of the Mn-ZnO are basically consistent with the diffraction peaks of the undoped ZnO, which is a typical hexagonal wurtzite structure. However, after doping Mn, the diffraction of the sample moves slightly to low angle, confirming that the Mn ion is doped into the lattice of ZnO, but still maintains the hexagonal phase structure.

[0082] Figure 5 The high-resolution XPS spectrum, a is the Mn2p spectrum of the Mn-ZnO nanoclusters prepared in Example 1, b is the O2s spectrum of the undoped ZnO prepared in the comparative experiment and the Mn-ZnO nanoclusters prepared in Example 1; from the figure Figure 5It can be seen that the valence state of Mn in Mn-ZnO is mainly +2 and +3. Due to the multiple valence states of Mn, it is beneficial to react with hydrogen peroxide and GSH, thereby exhibiting the characteristics of enzyme-like activity and GSH consumption. It can be seen from the XPS spectra of Mn 2p of Mn-ZnO that the Mn 2p3 / 2 peak can be divided into two peaks, which are respectively the peaks of Mn2+and Mn3+, and the peak of Mn2+is much stronger than that of Mn3+. The results show that the valence state of Mn in Mn-ZnO is mainly +2 and +3. Figure 5 It can be seen that the O2s XPS spectrum of Mn-ZnO can be divided into three absorption peaks, which are respectively the peaks of lattice oxygen (O L ), defect oxygen (O V ) and adsorbed oxygen (O C ), indicating that Mn doping leads to the existence of a large number of oxygen defects (oxygen vacancy concentration 33.3%).

[0083] Figure 6 The electron paramagnetic resonance spectra of undoped ZnO prepared in the comparative experiment and Mn-ZnO nanoclusters prepared in Example 1. It can be seen from the figure that Mn-ZnO has an obvious absorption peak at g = 2.002, which is a typical characteristic of oxygen defects, further confirming the existence of oxygen defects.

[0084] Figure 7 The low-temperature nitrogen adsorption-desorption isotherm and adsorption pore size distribution of Mn-ZnO nanoclusters prepared in Example 1, a is the low-temperature nitrogen adsorption-desorption isotherm, and b is the adsorption pore size distribution. It can be seen from the figure that Mn-ZnO has a typical IV-type adsorption isotherm, which is a typical characteristic of mesoporous materials. Figure 7 It can be seen from a that Mn-ZnO has a typical IV-type adsorption isotherm, which is a typical characteristic of mesoporous materials. Figure 7 It is shown that the pore size distribution of ZnO is mainly concentrated around 4 nm.

[0085] Figure 8 The piezoelectric phase diagram of undoped ZnO prepared in the comparative experiment and Mn-ZnO nanoclusters prepared in Example 1. It can be seen from the figure that ZnO and Mn-ZnO both exhibit a typical butterfly curve, which is a typical characteristic of piezoelectric materials. However, compared with undoped ZnO, the amplitude of Mn-ZnO is significantly larger. The calculation results show that the piezoelectric coefficient (d 33 = 44.1 pm / V) of Mn-ZnO is about 4 times that of ZnO (d 33 = 11.1 pm / V), indicating that Mn doping can significantly improve the piezoelectric performance of ZnO.

[0086] Figure 9The acoustic current curves of ZnO with different Mn doping amounts, 1 is undoped ZnO prepared in the comparative experiment, 2 is 1%-Mn-ZnO, 3 is 4%-Mn-ZnO, and 4 is 2%-Mn-ZnO; it can be seen from the figure that all the samples can generate current under the action of ultrasound, because the electrons on the valence band of Mn-ZnO or ZnO material are excited to the conduction band under the action of ultrasound, thereby forming the acoustic current. It can also be seen from the figure that when the Mn doping amount is 2%, the sample shows the maximum current intensity, indicating that 2% is the optimal doping amount. This is because the appropriate amount of Mn doping can form defects in the ZnO lattice, and the existence of oxygen defects can inhibit the recombination of electron-hole pairs, thereby showing the strongest current intensity. However, excessive doping will also lead to excessive oxygen defects, and excessive oxygen defects will act as a recombination center for electrons and holes, thereby leading to a decrease in the acoustic current intensity.

[0087] Figure 10 The electrochemical impedance of ZnO with different Mn doping amounts, 1 is undoped ZnO prepared in the comparative experiment, 2 is 1%-Mn-ZnO, 3 is 2%-Mn-ZnO, and 4 is 4%-Mn-ZnO; the size of the electrochemical impedance radius is an important parameter for measuring the charge separation ability, and the smaller the radius, the higher the charge separation efficiency. It can be seen from the figure that the radius of undoped ZnO is the largest, and the radius when the doping amount is 2% is the smallest, further confirming that 2%-Mn-ZnO has the highest charge separation efficiency.

[0088] Because diphenyl isobenzofuran (DPBF) is easily oxidized by active oxygen, which leads to a decrease in its absorbance at about 420 nm, therefore, the change in its absorbance can be monitored by a UV spectrophotometer to evaluate the piezocatalytic performance of the material. The specific test method is: 3 mg of ZnO or 2%-Mn-ZnO is dispersed in 3 mL of deionized water, 40 μL of DPBF / DMSO solution (10 mg / mL) is added, and then it is ultrasonicated (frequency 40 kHz) for a certain time and centrifuged, the supernatant is taken in a cuvette, and the absorbance at 420 nm is measured by UV-Vis; Figure 11The piezocatalytic performance of the prepared undoped ZnO and 2%-Mn-ZnO was evaluated by using DPBF as a probe. As can be seen from the figure, after the ZnO material was ultrasonically treated with the DPBF solution for a certain time, the absorbance of the DPBF decreased obviously. Notably, under the same conditions, the Mn-ZnO caused a more significant decrease in the absorbance of the DPBF, indicating that the Mn-ZnO had a stronger piezocatalytic performance than the ZnO. The catalytic mechanism is as follows: under the action of ultrasound, the electrons in the valence band of the Mn-ZnO are excited to the conduction band, and the electrons on the conduction band react with the oxygen in the solution to generate highly active superoxide anions; on the other hand, the holes on the valence band can oxidize water molecules into highly active hydroxyl radicals. Under the action of ultrasound, the superoxide anions and the hydroxyl radicals generated together react with the DPBF, resulting in a significant decrease in the absorbance of the DPBF. Because the Mn-ZnO material has a higher charge separation efficiency, it exhibits a stronger piezocatalytic performance.

[0089] The OXD activity of the Mn-ZnO nanoclusters prepared in Example One was studied under different pH conditions by using tetramethyl benzidine (TMB) as a probe. Because the colorless TMB can be oxidized by the superoxide ion into blue oxTMB, which has a strong absorption at about 650 nm, the OXD activity of the material can be evaluated by the color change of the TMB. The specific test method is as follows: 2 mg of the 2%-Mn-ZnO was dispersed in 2 mL of a NaAc-HAc (pH = 4.5) buffer solution, 40 μL of a TMB / DMSO solution (10 mg / mL) was added, and after a certain reaction time, the mixture was centrifuged, and the supernatant was detected by a UV-vis spectrophotometer at about 650 nm. Figure 12 The OXD activity and electron paramagnetic resonance spectrum of the 2%-Mn-ZnO under different pH conditions by using TMB as a probe, a is the OXD activity, and b is the electron paramagnetic resonance spectrum. As can be seen from the figure, the Mn-ZnO did not exhibit OXD activity under normal physiological conditions. With the decrease of the pH, the OXD activity became stronger and stronger. This indicates that the material can react with oxygen to generate highly toxic superoxide anions in the weakly acidic tumor microenvironment, thereby causing the apoptosis of tumor cells. Figure 12 b is the electron paramagnetic resonance spectrum of the sample, which can directly confirm the type of active oxygen generated. As can be seen, after the Mn-ZnO was reacted with the PBS solution for a certain time, the electron paramagnetic resonance spectrum exhibited a typical absorption peak of superoxide anions, and the intensity became stronger and stronger with the extension of time, further confirming the OXD activity of the Mn-ZnO.

[0090] The peroxidase activity (POD) of Mn-ZnO was studied by using TMB as a probe. The specific experimental method is as follows: 2 mg of 2%-Mn-ZnO was dispersed in 2 mL of NaAc-HAc (pH = 4.5) buffer solution, 40 μL of TMB / DMSO solution (10 mg / mL) and 20 μL of H2O2 solution (30% by mass) were added, and after a certain time of reaction, centrifugation was performed, and the supernatant was detected by UV-vis spectrophotometer at about 650 nm. Figure 13 As a probe, the peroxidase activity of 2%-Mn-ZnO and the electron paramagnetic resonance spectrum are shown in FIG. 2a and FIG. 2b. The results show that Mn-ZnO can effectively catalyze hydrogen peroxide to generate hydroxyl radicals, which confirms the POD enzyme catalytic activity of Mn-ZnO. Therefore, by using the POD activity of Mn-ZnO, the hydrogen peroxide in the tumor microenvironment can be converted into highly toxic hydroxyl radicals, thereby causing tumor cell apoptosis.

[0091] The consumption behavior of GSH by Mn-ZnO was studied by using 5,5-dithiobisnitrobenzoic acid (DTNB) as a probe. The specific test method is as follows: 2.0 mg of 2%-Mn-ZnO was added to 2 mL of GSH aqueous solution with a concentration of 2 mM, and after a certain time of reaction, centrifugation was performed, 100 μL of the supernatant was mixed with 500 μL of DTNB solution (0.2 mmol / L), and the absorbance at about 410 nm was detected by UV-vis. Figure 14 The consumption performance of 2%-Mn-ZnO on glutathione (GSH) is shown in FIG. 3. As can be seen from the figure, the content of GSH gradually decreases due to Mn-ZnO, which indicates that Mn-ZnO can effectively consume GSH. This is because GSH with reducing property can undergo redox reaction with Mn 3+ in Mn-ZnO due to oxidation-reduction reaction. Figure 14 As can be seen from FIG. 3b, under the action of ultrasound (1.0 MHz, 1.0 W / cm 2 ), the consumption ability of Mn-ZnO on GSH is further enhanced, which indicates that ultrasound can promote the consumption of GSH by Mn-ZnO.

[0092] Firstly, 4T1 cells were planted in a 96-well plate, and incubated at 37°C under 5% CO2 for 24 h until the cell monolayer covered the bottom of the well. Then, 0.1 mL of 2%-Mn-ZnO solution with different concentrations was added, and incubated at 37°C under 5% CO2 for 24 h. Then, 20 μL of MTT solution (0.5% MTT) was added to each well, and incubated for another 4 h. The culture medium was removed, 150 μL of dimethyl sulfoxide was added to each well, and the absorbance of each well was measured at 490 nm by an enzyme-labeled instrument. Figure 15For the MTT method to evaluate the killing column of 2%-Mn-ZnO on 4T1 cells, 1 is the blank control group, 2 is the simple ultrasound group, 3 is 2%-Mn-ZnO, 4 is 2%-Mn-ZnO+ultrasound. It can be seen that the simple ultrasound has little effect on the activity of 4T1 cells, indicating that the effect of ultrasound on cells can be ignored. Mn-ZnO material has a certain killing ability on 4T1 cells, and the cell viability decreases gradually with the increase of concentration, which shows the enzyme catalytic activity of Mn-ZnO. However, under the action of ultrasound, Mn-ZnO shows stronger cell killing ability, which is due to the production of more active oxygen by Mn-ZnO under the action of ultrasound.

[0093] With APF and DHE as probes, hydroxyl radicals and superoxide anions produced in 4T1 cells were selectively discriminated. After proliferation, the cells were incubated with 2%-Mn-ZnO solution (100 μg / mL) in a cell incubator at 37°C for 4 h, and the cells were washed with PBS for 2-3 times, then specific probes APF or DHE (10 μM) were added and stained for 20 min. Then, the cells were irradiated with a handheld ultrasound instrument (1 MHz, 1.0 W / cm 2 ) for 1 min, and the fluorescence photos of the cells were taken by a laser confocal microscope. Figure 16 The laser confocal photos of 2%-Mn-ZnO after incubation with 4T1 cells. As can be seen from the figure, when Mn-ZnO is incubated with 4T1 cells for 4 h and stained with APF, 4T1 cells show bright green fluorescence, indicating that Mn-ZnO reacts with peroxidase in 4T1 cells to generate hydroxyl radicals, showing the catalytic performance of POD enzyme activity. In addition, Mn-ZnO is incubated with 4T1 cells and stained with DHE, and the cells show bright red, indicating that superoxide anions are generated, showing the catalytic activity of OXD enzyme. Importantly, under the action of ultrasound, 4T1 cells show stronger green and red fluorescence signals, respectively, indicating that Mn-ZnO produces more active oxygen under the action of ultrasound, which is due to the good piezoelectric property of Mn-ZnO.

[0094] 4T1 cells were inoculated in a 12-well plate at a density of 1×105 cells per well, and incubated overnight. Then 2%-Mn-ZnO solution (100 μg / mL) was added, and then incubated in the incubator for 6 hours. Subsequently, 4T1 cells were irradiated with ultrasound (1 MHz, 1.0 W / cm 2 ) for 1 min, and annexin V-FITC / PI apoptosis detection kit was used for quantification of apoptotic cells. Figure 17The image shows a flow cytometry plot of 4T1 cells. As can be seen, Mn-ZnO material effectively induced apoptosis in 4T1 tumor cells, with approximately 29.5% of cells undergoing apoptosis. This is attributed to the enzymatic catalytic activity of Mn-ZnO material. Further ultrasound irradiation showed a significant increase in the apoptosis rate of 4T1 cells, reaching 67.2%, indicating that ultrasound can significantly enhance tumor cell apoptosis.

[0095] Mn-ZnO was used for tumor treatment in tumor-bearing mice: Twenty female BALB / c mice bearing 4T1 cells were randomly divided into four groups (n=5 per group): a control group, an ultrasound group, a Mn-ZnO group, and a Mn-ZnO + ultrasound group. In the Mn-ZnO + ultrasound group, when the tumor volume reached approximately 60 mm², tumors were treated. 3 At that time, a 2% Mn-ZnO solution (1 mg / mL) was injected into mice via the tail vein (the dose of 2% Mn-ZnO was 15 mg / kg). Twelve hours after injection, the mice were treated with an ultrasound therapy device (1 MHz, 1.0 W / cm²). -2 The tumor site was subjected to ultrasound treatment for 3 minutes. The treatment method was similar for the Mn-ZnO group and the Mn-ZnO + ultrasound group, except that the Mn-ZnO group did not receive ultrasound treatment. For the ultrasound group, mice were injected with an equal volume of physiological saline via the tail vein, and the tumor site was irradiated with ultrasound. The treatment cycle was 14 days, with treatment performed on the first and seventh days. The mouse's weight and tumor volume were recorded every two days. After 14 days of treatment, the mice were sacrificed, and the tumor and major organs were collected for H&E and TUNEL staining analysis. Figure 18 The figures show the changes in tumor volume and photographs of the tumors in mice after treatment. Figure a shows the change in tumor volume, and figure b shows a photograph of the tumor. As can be seen from the figures, after injecting Mn-ZnO material into mice via the tail vein (solute 2% - Mn-ZnO dosage: 15 mg / kg), and following two ultrasound treatments (day 1 and day 7), tumor growth in the mice was significantly inhibited after 14 days, demonstrating that the material has a good therapeutic effect on tumors.

[0096] Figure 19 Blood routine thermograms of healthy mice after injection of 2% Mn-ZnO solution (1 mg / mL). As shown in the figure, after injection of Mn-ZnO via the tail vein (dose of 2% Mn-ZnO: 20 mg / kg), the blood routine indicators of healthy mice did not change significantly, indicating that the material has good biocompatibility.

[0097] Specific method: 1 mg of 2% Mn-ZnO was dispersed in 5 mL of phosphate buffer solution with different pH values ​​(pH = 5.5 or 7.4) and stirred at 37℃ for 2 days. 1 mL of the mixed solution was taken at 12h, 24h, and 48h, ultrasonically dispersed, and then used for TEM testing to observe the morphological changes of the material. Figure 20Degradation of 2%-Mn-ZnO in different acidic conditions. From the figure, it can be seen that Mn-ZnO has obvious degradation behavior in weak acid solution of pH 5.5, and after 48 h, it basically becomes smaller nanoparticles and thin slices. On the contrary, the morphology of Mn-ZnO in the phosphate buffer solution of pH 7.4 changes little.

[0098] After the healthy mice were injected with 2%-Mn-ZnO solution (1 mg / mL) through the tail vein (the dose of solute 2%-Mn-ZnO: 20 mg / kg), the tumor-bearing mice were placed in a metabolic cage, and urine and feces were collected regularly. Then the content of Zn in the feces and urine was determined by inductively coupled plasma optical emission spectrometer (ICP-OES); Figure 21 The content of zinc ion in the feces and urine of mice. From the figure, it can be seen that Zn ion is mainly excreted by feces through in vivo metabolism, so it can greatly reduce the risk of Zn accumulation in vivo and reduce the long-term biological toxicity of nanomaterials.

[0099] Figure 22 The infrared spectrum of 2%-Mn-ZnO. From the figure, it can be seen that the absorption peak of Mn-ZnO at 3410 cm -1 nearby can be attributed to the absorption of hydroxyl, while the absorption at 2966, 2910 and 1398 cm -1 indicates the presence of C-H groups on the surface of Mn-ZnO. The absorption at 1606 and 1072 cm -1 can be attributed to the absorption of carboxyl and the stretching vibration of Zn-O, respectively. Therefore, the FT-IR results show that there are abundant hydrophilic hydroxyl and carboxyl groups on the surface of Mn-ZnO.

Claims

1. A method for preparing biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity, characterized by It is carried out according to the following steps: I. Under the condition that the temperature is 40-60℃, Zn(NO3)2·6H2O, Mn(NO3)2 and diethylene glycol are stirred and mixed uniformly to obtain a mixed solution; The mass ratio of Zn(NO3)2·6H2O to Mn(NO3)2 is 1:(0.02-0.12); the mass of Zn(NO3)2·6H2O to the volume of diethylene glycol is 1g:(60-80)mL; II. Under the condition that the stirring speed is 300-500rpm / min, the mixed solution is heated to 210-230℃ at a heating rate of 1-2℃ / min, and heated to reflux at 210-230℃ for 10-14h, and cooled to room temperature to obtain a brownish suspension; III. Anhydrous ethanol is added to the brownish suspension, and then the supernatant is removed by centrifugation to obtain a precipitate, which is washed, dried and ground in sequence to complete the preparation method of the biodegradable Mn-ZnO nanocluster with porous structure and enzyme-like activity.

2. The method for preparing biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity according to claim 1, characterized in that In step I, Zn(NO3)2·6H2O, Mn(NO3)2 and diethylene glycol are stirred for 20-40min at a temperature of 40-60℃ and a stirring speed of 300-500rpm / min.

3. The method for preparing biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity according to claim 1, characterized in that... In step III, the volume ratio of the brownish suspension to anhydrous ethanol is 1:(1-1.5).

4. The method for preparing biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity according to claim 1, characterized in that... In step III, the supernatant is removed by centrifugation at a speed of 80000-10000r / min for 8-12min.

5. The method for preparing biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity according to claim 1, characterized in that... In step III, the washing is carried out 2-3 times with anhydrous ethanol and then 2-3 times with deionized water.

6. The method for preparing biodegradable Mn-ZnO nanoclusters with porous structure and enzyme-like activity according to claim 1, characterized in that... In step III, the drying is carried out at a temperature of 40-50℃ under vacuum for 10-14h.

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