CN113679838B - Vanadium nano-enzyme and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a vanadium nano-enzyme and a preparation method and application thereof, wherein the preparation method comprises the following steps: dispersing vanadium powder in an organic solvent, performing cyclic ultrasonic treatment under ice bath conditions, centrifuging, and collecting precipitate to obtain a vanadium nanomaterial; preparing a vanadium nano material into a dispersion liquid, then adding amphiphilic molecules into the dispersion liquid under the ultrasonic condition, continuing ultrasonic treatment, stirring, reacting, centrifuging, and collecting precipitate to obtain the vanadium nano enzyme. The raw materials required by the preparation method for preparing the vanadium nano enzyme are wide in sources, the preparation method is simple and the size is controllable; the vanadium nano enzyme provided by the invention has the advantages of good biocompatibility and biodegradability, and has good photo-thermal effect and catalytic activity, and experiments prove that the vanadium nano enzyme can obviously kill and remove tumor cells by cooperating with the photo-thermal effect and the catalytic activity, so that the effect of treating tumor diseases is achieved.
Description
Technical Field
The invention belongs to the technical field of biological medicine. More particularly, relates to a vanadium nano-enzyme and a preparation method and application thereof.
Background
Photothermal therapy is a treatment method that uses a material with high photothermal conversion efficiency, injects it into the inside of a human body, gathers near tumor tissue using a targeting recognition technique, and converts light energy into heat energy under irradiation of an external light source (typically near infrared light) to kill cancer cells. The common photo-thermal materials comprise noble metals such as gold Au, silver Ag, platinum Pt and the like, and the photo-thermal conversion efficiency is high. For example, chinese patent application CN101711872a discloses a nano gold nanomaterial, which selects gold as a research content, has a quasi-spherical shell shape and a hollow structure inside, and can not only raise the temperature in a local range after near infrared laser irradiation, be used together with an antitumor drug to kill tumor cells, but also be used as a developer in an infrared tomography technology. However, the main material adopted by the material is gold, and the raw material has high price and high cost; on the other hand, the anti-tumor effect achieved by using the photo-thermal material gold alone is limited, the tumor cells can not be completely removed, and the anti-tumor drugs are combined to kill the tumor cells better. Therefore, there is an urgent need to provide a material having a remarkable killing and clearing effect on tumor cells.
Disclosure of Invention
The invention aims to overcome the defects of high cost and limited tumor removal effect of the existing anti-tumor photo-thermal material, and provides the vanadium nano-enzyme which has low cost, has a catalytic effect and can significantly remove tumor cells.
The invention aims to provide a preparation method of vanadium nano-enzyme.
The invention also aims to provide an application of the vanadium nano-enzyme in preparing a medicine for preventing and treating tumors.
The invention also aims to provide the application of the vanadium nano-enzyme as a catalyst in catalyzing oxidation-reduction reaction.
The above object of the present invention is achieved by the following technical scheme:
the first aspect of the invention provides a preparation method of vanadium nanoenzyme, which comprises the following steps:
s1, dispersing vanadium powder in an organic solvent, performing cyclic ultrasonic treatment under ice bath conditions, centrifuging, and collecting precipitate to obtain a vanadium nanomaterial;
s2, preparing a vanadium nano material into a dispersion liquid, then adding amphiphilic molecules into the dispersion liquid under the ultrasonic condition, continuing ultrasonic treatment, stirring, reacting, centrifuging, and collecting precipitate to obtain the vanadium nano enzyme.
Further, in step S2, the amphiphilic molecule is phospholipid-polyethylene glycol, polyvinylpyrrolidone or hydroxyethyl starch.
Further, in step S1, the organic solvent is N-methylpyrrolidone, dimethylformamide or dimethylsulfoxide.
Further, in step S1, the cyclic ultrasonic treatment is performed under a power condition of 100-1000W with an on/off period: 2-8 s/2-8 s is circulation, and the ultrasonic treatment is carried out for 12-72 h. Preferably, the conditions of the cyclic ultrasonic treatment are at 700W power conditions with on/off cycles: 5s/5s is circulation, and ultrasonic treatment is carried out for 48 hours; the centrifugation is that the first centrifugation is carried out at 3000-8000 rpm/min, the supernatant is collected, and the second centrifugation is carried out at 10000-50000 rpm/min; preferably, the first centrifugation time is 5-20 min, and the second centrifugation time is 15-30 min.
Further, in the step S1, the mass volume ratio of the vanadium powder to the organic solvent is (0.1-10) g: (50-200) mL. Preferably, the mass volume ratio of the vanadium powder to the organic solvent is (0.1-5) g: (50-100) mL; more preferably, the mass volume ratio of the vanadium powder to the organic solvent is 1g:100mL.
Further, in step S2, the mass ratio of the vanadium nanomaterial to the amphiphilic molecule is 1: (0.5-4). Preferably, the mass ratio of the vanadium nanomaterial to the amphiphilic molecule is 1: (0.5-2); more preferably, the mass ratio of the vanadium nanomaterial to the amphiphilic molecule is 1:2; the centrifugal speed is 10000-50000 rpm/min.
Preferably, in the step S2, the concentration of the vanadium nanomaterial in the vanadium nanomaterial dispersion is 0.5-4 mg/mL, and the solvent is ethanol; the amphiphilic molecules are added into the dispersion liquid, namely amphiphilic molecule solution is added into the dispersion liquid, the concentration of the amphiphilic molecules in the amphiphilic molecule solution is 0.5-4 mg/mL, and the solvent is an acetone-ethanol mixed solution (acetone: ethanol=2:3).
Preferably, in step S2, the stirring reaction is performed by using a magnetic stirrer, and the stirring speed is 600-1500 rpm, and the stirring is performed overnight.
Preferably, in step S2, the method further comprises washing the collected precipitate, wherein the solvent for washing is ethanol.
Further, in step S2, the amphiphilic molecules are added to the dispersion liquid under the ultrasonic condition and the ultrasonic treatment is continued for a period of time in an ice bath under the power condition of 100-500W.
Furthermore, the invention also provides the vanadium nano-enzyme prepared by the preparation method. Vanadium in the vanadium nano enzyme is one or more of pentavalent, tetravalent and trivalent; the average grain diameter of the obtained vanadium nano enzyme is 50-800 nm.
In addition, the invention also provides application of the vanadium nano-enzyme in preparing a medicine for preventing and treating tumors.
Preferably, the medicine can be combined with photothermal therapy to treat tumors, so that better tumor removal and treatment effects can be achieved.
In addition, the invention also provides application of the vanadium nano-enzyme serving as a catalyst in catalytic oxidation-reduction reaction. Research proves that the vanadium nano-enzyme serving as a catalyst has the activity of one or more enzymes of peroxidase-like enzyme, horseradish peroxidase-like enzyme, oxidase-like enzyme, superoxide dismutase-like enzyme, glutathione oxidase-like enzyme and catalase-like enzyme, and is an excellent natural enzyme substitute.
The vanadium nano enzyme has peroxidase-like activity, and can catalyze hydrogen peroxide (H) 2 O 2 ) Generate a large amount of hydroxyl radicals (OH) to kill tumor cells; has glutathione oxidase-like activity, and can catalyze reduced Glutathione (GSH) to be changed into oxidized glutathione; has catalase-like activity and can catalyze H in tumor microenvironment 2 O 2 Generating oxygen; the vanadium nano-enzyme with various enzyme activities can form a nano-platform for self-cascade regulation and control of tumor microenvironment, generate a large amount of OH and deplete GSH in cells, thereby achieving the effect of promoting apoptosis of tumor cells. Meanwhile, the vanadium nano enzyme has a photo-thermal effect, can further cauterize tumors by combining photo-thermal treatment, enhances nano enzyme activity, and realizes excellent tumor treatment effect by synergistic effect.
The invention has the following beneficial effects:
the invention provides a vanadium nano enzyme, which has the advantages of wide sources of required raw materials, simple preparation method and controllable size; the vanadium nano enzyme provided by the invention has the advantages of good biocompatibility and biodegradability, and has good photo-thermal effect and catalytic activity, and experiments prove that the vanadium nano enzyme can obviously kill and remove tumor cells by cooperating with the photo-thermal effect and the catalytic activity, so that the effect of treating tumor diseases is achieved.
Drawings
FIG. 1 is an electron microscope scan of the vanadium nanoenzyme obtained in example 1 of the present invention.
FIG. 2 is a mapping chart of the elemental distribution analysis of the vanadium nanoenzyme obtained in example 1 of the present invention.
FIG. 3 is an X-ray photoelectron spectrum of the vanadium nanoenzyme and vanadium powder obtained in example 1 of the present invention.
FIG. 4 is an X-ray diffraction pattern of the vanadium nanoenzyme and vanadium powder obtained in example 1 of the present invention.
FIG. 5 is an ultraviolet-visible spectrum of the vanadium nanoenzyme obtained in example 1 of the present invention.
FIG. 6 is a graph showing the study statistics of the photo-thermal properties of the vanadium nanoenzyme obtained in example 1 of the present invention.
FIG. 7 is a graph showing the study statistics of the enzyme activity of the vanadium nanoenzyme obtained in example 1 of the present invention; wherein, the A-type catalase, the B-type peroxidase and the C-type glutathione oxidase.
FIG. 8 is a graph showing experimental study statistics of cytotoxicity of vanadium nanoenzyme tumor obtained in example 1 of the present invention; a-single enzyme catalysis treatment and B-enzyme catalysis-photo-thermal cooperative treatment.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1
The embodiment is a preparation method of vanadium nano-enzyme, which comprises the following steps:
s1, 1g of vanadium powder is weighed and dispersed in 100mL of N-methylpyrrolidone (NMP), 700W is carried out under the ice bath condition, and the on/off period is as follows: performing probe ultrasonic treatment for 48h in a circulating way for 5s/5s, centrifuging for 10min at 5000rpm/min by using a low-temperature ultracentrifuge, removing non-stripped vanadium crystals, collecting supernatant, centrifuging for 20min at 20000rpm/min, and collecting precipitate to obtain vanadium nanomaterial;
s2, preparing the vanadium nanomaterial obtained in the step S1 and ethanol into a vanadium nanomaterial dispersion liquid with the concentration of 1mg/mL, dropwise adding 1mL of distearoyl phosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) solution (2 mg/mL, wherein the solvent is acetone: ethanol=2:3) into the vanadium nanomaterial dispersion liquid with the concentration of 1mg/mL under the ultrasonic condition of ice water bath 300W, continuing ultrasonic treatment for 30min, magnetically stirring for overnight reaction at 600-1500 rpm, centrifuging at 20000rpm for 20min after the reaction is finished, and washing with ethanol twice to obtain the vanadium nanomaterial.
Example 2
The embodiment is a preparation method of vanadium nano-enzyme, which comprises the following steps:
s1, 1g of vanadium powder is weighed and dispersed in 100mL of dimethylformamide, 1000W is carried out under the ice bath condition, and the on/off period is carried out: performing probe ultrasonic treatment for 12h in a circulating way for 8s/8s, centrifuging for 5min at 8000rpm/min by using a low-temperature ultracentrifuge, removing non-stripped vanadium crystals, collecting supernatant, centrifuging for 15min at 50000rpm/min, and collecting precipitate to obtain vanadium nanomaterial;
s2, preparing the vanadium nanomaterial obtained in the step S1 and ethanol into a vanadium nanomaterial dispersion liquid with the concentration of 1mg/mL, dropwise adding 1mL of distearoyl phosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) solution (2 mg/mL, wherein the solvent is acetone: ethanol=2:3) into the vanadium nanomaterial dispersion liquid with the concentration of 1mg/mL under the ultrasonic condition of ice water bath 300W, continuing ultrasonic treatment for 30min, magnetically stirring for overnight reaction at 600-1500 rpm, centrifuging at 20000rpm for 20min after the reaction is finished, and washing with ethanol twice to obtain the vanadium nanomaterial.
Example 3
The embodiment is a preparation method of vanadium nano-enzyme, which comprises the following steps:
s1, 1g of vanadium powder is weighed and dispersed in 100mL of dimethyl sulfoxide, 300W is carried out under the ice bath condition, and the on/off period is carried out: performing probe ultrasonic treatment for 72h in a circulating way for 2s/2s, centrifuging for 20min at 3000rpm/min by using a low-temperature ultracentrifuge, removing non-stripped vanadium crystals, collecting supernatant, centrifuging for 20min at 25000rpm/min, and collecting precipitate to obtain vanadium nanomaterial;
s2, preparing the vanadium nanomaterial obtained in the step S1 and ethanol into a vanadium nanomaterial dispersion liquid with the concentration of 1mg/mL, dropwise adding 1mL of distearoyl phosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) solution (2 mg/mL, wherein the solvent is acetone: ethanol=2:3) into the vanadium nanomaterial dispersion liquid with the concentration of 1mg/mL under the ultrasonic condition of ice water bath 300W, continuing ultrasonic treatment for 30min, magnetically stirring for overnight reaction at 600-1500 rpm, centrifuging at 20000rpm for 20min after the reaction is finished, and washing with ethanol twice to obtain the vanadium nanomaterial.
The structural characteristics and performance of the vanadium nanoenzyme prepared in example 1 are tested in the following, and similar effects can be achieved in other examples.
Test example 1 determination of physicochemical Properties of vanadium nanoenzyme
The vanadium nanoenzyme obtained in example 1 was subjected to electron microscopy scanning analysis, and the result is shown in fig. 1. The graph shows that the polyethylene glycol modified vanadium nano-enzyme obtained in the embodiment 1 has good dispersibility and the particle size is between 180 and 220 nm.
The vanadium nanoenzyme obtained in example 1 was subjected to element distribution analysis, and mapping spectra were obtained, and the results are shown in fig. 2. The graph shows that V, C, N, O elements exist in the polyethylene glycol modified vanadium nano-enzyme, which indicates that the vanadium nano-enzyme provided by the invention is successfully prepared.
X-ray photoelectron spectroscopy and X-ray diffraction pattern analysis were performed on the vanadium nanoenzyme and vanadium powder obtained in example 1, respectively, and the results are shown in FIGS. 3 to 4. The graph shows that V, C, N, O elements exist in the polyethylene glycol modified vanadium nano-enzyme and the vanadium powder, so that the vanadium nano-enzyme is successfully prepared and does not dope byproducts; the characteristic absorption peak of the vanadium nano-enzyme is completely matched with the characteristic peak of the vanadium powder, which shows that the vanadium nano-enzyme is successfully prepared and the crystal structure is unchanged.
Experimental example 2 determination of optical Properties of vanadium nanoenzyme
The optical absorption of the vanadium nanoenzyme obtained in example 1 was evaluated, and the results are shown in fig. 5. From the graph, the polyethylene glycol modified vanadium nano enzyme has strong optical absorption in ultraviolet, visible light and near infrared regions, and shows concentration dependence.
The photo-thermal performance of the vanadium nano-enzyme modified by polyethylene glycol is evaluated under the condition of 808nm wavelength laser, and the result is shown in figure 6.
From the graph 6A, the vanadium nano-enzyme modified by polyethylene glycol has higher temperature rise under 808nm laser excitation and excellent photo-thermal performance; from fig. 6B, it can be seen that the photo-thermal stability of the polyethylene glycol modified vanadium nanoenzyme is good. Therefore, the polyethylene glycol modified vanadium nano-enzyme prepared by the invention has the required temperature for cauterizing tumors by photothermal treatment.
Experimental example 3 determination of catalytic Property of vanadium nanoenzyme enzyme
The catalase-like activity of the vanadium nano-enzyme obtained in example 1 was tested by the following method: different concentrations of vanadium nanoenzymes (0, 12.5, 25, 50 μg/mL) were dispersed in phosphate buffer (pH 7.4), once H was added with a syringe 2 O 2 (10 mM), i.e., measuring solution absence with JPSJ-605F portable dissolved oxygen meterThe oxygen concentration at the same time point is shown in FIG. 7A.
The vanadium nanoenzyme obtained in example 1 was tested for peroxidase-like activity by the following method: dispersing vanadium nanoenzymes (0, 12.5, 25, 50, 100 mug/mL) with different concentrations in phosphate buffer (pH 7.4), then adding 10 mug/mL methylene blue indicator, finally adding H 2 O 2 (10 mM) the solution was measured for UV absorption at various time points by UV-visible spectroscopy, and the results are shown in FIG. 7B.
The vanadium nanoenzyme obtained in example 1 was tested for glutathione oxidase-like activity by the following method: different concentrations of vanadium nanoenzymes (0, 12.5, 25, 50, 100 μg/mL) were dispersed in phosphate buffer (ph 7.4), followed by the addition of 0.1mM GSH, and finally the consumption of GSH was detected using a 5,5' -dithiobis (2-nitrobenzoic acid) probe, see figure 7C for results.
As can be seen from fig. 7A, the polyethylene glycol modified vanadium nano-enzyme has strong oxygen generating capability in hydrogen peroxide environment, has concentration dependence, and shows strong catalase-like activity; as can be seen from fig. 7B, the polyethylene glycol modified vanadium nano-enzyme has remarkable OH generating capability and concentration dependence, and shows stronger peroxidase-like activity; from fig. 7C, it can be seen that the polyethylene glycol modified vanadium nano-enzyme has obvious GSH oxidation capability, can reduce the anti-oxidation capability of tumor, and shows stronger glutathione oxidase-like activity. In conclusion, the polyethylene glycol modified vanadium nano enzyme has the capabilities of hydrogen peroxidase-like, peroxidase-like and glutathione oxidase-like, and has the capabilities of controlling tumor microenvironment to kill tumors and preventing tumor recurrence and metastasis.
Experimental example 4 determination of the Activity of vanadium nanoenzyme on tumor cells
The vanadium nanoenzyme obtained in example 1 was tested for toxicity induced by enzyme catalysis of human breast cancer cell MCF-7 by the following method: MCF-7 cells were plated at 8X 10 per well 3 The density of individual cells was seeded in 96-well plates and cultured for 12 hours. After washing once with PBS, cells were incubated with gradient concentrations (0, 3.13, 6.25, 12.5, 25, 50. Mu.g/mL) of vanadium nanoenzymes for 12 or 24 hours, respectively. Washing with PBSAfter incubation for 2 hours using Cell Counting Kit-8 (CCK-8), absorbance was measured at a wavelength of 450nm and standard cell viability assays were performed to determine relative cell viability, see FIG. 8A for results.
The vanadium nanoenzyme obtained in example 1 tests toxicity caused by enzyme catalysis-photo-thermal synergy of human breast cancer cells MCF-7, and the testing method comprises the following steps: MCF-7 cells were plated at 8X 10 per well 3 The density of individual cells was seeded in 96-well plates and cultured for 12 hours. After washing once with PBS, the cells were incubated with gradient concentrations (0, 3.13, 6.25, 12.5, 25, 50. Mu.g/mL) of vanadium nanoenzymes for 6 hours, followed by 808nm laser irradiation (1W/cm) 2 5 min). After further incubation for 18 hours, washing with PBS, incubation with Cell Counting Kit-8 (CCK-8) for 2 hours, absorbance was measured at a wavelength of 450nm and standard cell viability assays were performed to determine relative cell viability, see FIG. 8B for results.
As can be seen from fig. 8A, under single enzyme catalytic treatment, the polyethylene glycol modified vanadium nanoenzyme exhibits a certain toxicity to tumor cells and concentration dependence; as can be seen from fig. 8B, under the synergistic treatment of photo-thermal and enzyme catalysis, the toxicity of the polyethylene glycol modified vanadium nano-enzyme to tumor cells is greatly enhanced, and a remarkable tumor killing effect is achieved.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. The preparation method of the vanadium nano-enzyme is characterized by comprising the following steps of:
s1, dispersing vanadium powder in an organic solvent, performing cyclic ultrasonic treatment under ice bath conditions, centrifuging, and collecting precipitate to obtain a vanadium nanomaterial, wherein the organic solvent is N-methylpyrrolidone, the centrifuging is that the first centrifuging is performed at 3000-8000 rpm/min, the supernatant is collected, and the second centrifuging is performed at 10000-50000 rpm/min;
s2, preparing a vanadium nano material into a dispersion liquid, then adding amphiphilic molecules into the dispersion liquid under the ultrasonic condition, continuing ultrasonic treatment, stirring, reacting, centrifuging, and collecting precipitate to obtain the vanadium nano enzyme, wherein the amphiphilic molecules are phospholipid-polyethylene glycol.
2. The method according to claim 1, wherein in step S1, the cyclic ultrasonic treatment is performed at a power of 100 to 1000W with on/off cycles: 2-8 s/2-8 s is circulation, and the ultrasonic treatment is carried out for 12-72 h.
3. The preparation method according to claim 1, wherein in the step S1, the mass-to-volume ratio of the vanadium powder to the organic solvent is (0.1-10) g: (50-200) mL.
4. The preparation method according to claim 1, wherein in step S2, the mass ratio of the vanadium nanomaterial to the amphiphilic molecule is 1: (0.5-4); the centrifugal speed is 10000-50000 rpm/min.
5. A vanadium nanoenzyme prepared by the preparation method of any one of claims 1 to 4.
6. The use of the vanadium nanoenzyme according to claim 5 for preparing a medicament for preventing and treating tumors.
7. Use of the vanadium nanoenzyme according to claim 5 as a catalyst for catalytic redox reactions for non-therapeutic purposes.
8. The use according to claim 7, wherein the vanadium nanoenzyme has activity of one or more enzymes selected from the group consisting of peroxidase-like, glutathione oxidase-like and catalase-like as a catalyst.
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