CN115137820A

CN115137820A - Preparation method of nano enzyme compound for treating tumors

Info

Publication number: CN115137820A
Application number: CN202210662888.XA
Authority: CN
Inventors: 卢晓英; 宋华军; 江奇; 吴文帝; 张兵
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2022-06-13
Filing date: 2022-06-13
Publication date: 2022-10-04
Anticipated expiration: 2042-06-13
Also published as: CN115137820B

Abstract

The invention discloses a preparation method of a nano enzyme compound for treating tumors, which comprises the following steps: ball-milling CNT and steel ball; dispersing ball-milled CNT; sequentially adding urea and sodium dodecyl sulfate into a beaker, and adding the dispersed CNT solution; dissolving ferric sulfate in water and adding the solution; dissolving phosphoric acid in water and adding the solution; heating, cooling and filtering the mixed solution to obtain FePOs and CNT composites; introducing H2 into the FePOs and CNT composite for high-temperature calcination to obtain FeP and CNT composite; weighing FeP, CNT compound and ionic surfactant for grinding; dissolving another ionic surfactant in water, adding the ground sample, dispersing, standing, and taking the upper-layer dispersion liquid; dissolving the prepared GO liquid in water and then dispersing; transporting the CNT dispersion to GO liquid, and carrying out electrostatic self-assembly; dissolving ammonium bicarbonate in water, adding the mixture into the mixed solution, performing electrostatic self-assembly and then performing suction filtration to obtain a FeP, CNT and GO compound.

Description

Preparation method of nano enzyme compound for treating tumors

Technical Field

The invention relates to the technical field of nano biology, in particular to a preparation method of a nano enzyme compound for treating tumors.

Background

With the emergence of nano-enzyme with enzyme-like activity, the enzyme therapy anti-tumor will have the opportunity to make breakthrough progress in practical application, the nano-enzyme with peroxidase activity, catalase activity, oxidase activity and superoxide dismutase activity is widely used in the field of tumor therapy, and in the tumor therapy, the conventional physical means can also play an effective killing role on tumor cells, but the side effect is large, and researches show that the tumor cells can be induced to die by relatively mild stimulation (sound, light, electricity, magnetism and the like).

However, the research on treating tumors by nano-enzyme is relatively independent at present, and few researches are conducted to explore the anti-tumor effect of nano-enzyme under the condition of external stimulation, carbon nano-tubes (carbon nano-tubes, CNTs) and Graphene (GO) are nano-materials with enzyme-like activity and have certain electrical property, magnetic property and photothermal property, carbon-based nano-enzyme has certain effect on resisting tumors, but the application of the carbon-based nano-enzyme in tumor treatment is limited due to low enzymatic activity of the carbon-based nano-enzyme, and iron-based nano-enzyme generally has high enzyme-like activity and has good effect on tumor treatment, so that the iron-based nano-material and the carbon-based nano-material are combined to form a nano-enzyme compound which can enhance the enzymatic activity and respond to external stimulation.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a preparation method of a nano-enzyme compound for treating tumors, which can realize the preparation of the nano-enzyme compound of FePOs, CNTs and GO or the nano-enzyme compound of FeP, CNT and GO with various enzyme activities and multi-stimulus response, can realize the combination of various stimulus conditions and enzyme activities with anti-bone tumor activity, reveal the molecular biological mechanism of nano-enzyme under the synergistic action of stimulus and enzyme activities, and lay the theoretical basis for stimulating the combination of nano-enzyme with anti-tumor activity.

The technical scheme of the invention is as follows: a preparation method of a nano enzyme compound for treating tumors, wherein the nano enzyme compound is a compound of FePOs, CNTs and GO or a compound of FeP, CNTs and GO, and comprises the following steps:

the method comprises the following steps: putting CNT and steel ball into a ball milling tank according to the mass ratio of 45-55:0.5-1.5, adding absolute ethyl alcohol to immerse the steel ball, carrying out ball milling for 3-5h, taking out the CNT, cleaning with absolute ethyl alcohol and deionized water, and drying for later use;

step two: taking 45-55mgCNT subjected to ball milling and drying in the step one, adding 45-55ml of deionized water, and performing ultrasonic dispersion for 0.5-1.5h for later use;

step three: taking a beaker, sequentially adding 2-4g of urea, 0.2-0.3g of sodium dodecyl sulfate and 32-36ml of deionized water into the beaker, fully dissolving, then adding 40-60ml of CNT solution dispersed in the step two, and uniformly stirring;

step four: weighing 0.05-0.15g of ferric sulfate, dissolving in 6-10ml of deionized water, slowly dripping into the uniformly stirred solution in the step three, and continuously stirring for 15-25min;

step five: weighing 0.2-0.3g of phosphoric acid, dissolving in 6-10ml of deionized water, slowly dripping the solution uniformly stirred in the step four, and continuously stirring for 15-25min;

step six: putting the mixed solution uniformly stirred in the step five into a reaction kettle, heating to 135-145 ℃, carrying out hydrothermal reaction for 1.5-2.5h, cooling to room temperature, carrying out suction filtration, washing for 2-4 times by using deionized water and absolute ethyl alcohol to obtain a FePOs and CNT compound, drying at 55-65 ℃, and putting into a drying tower for later use;

step seven: placing the FePOs and CNT composite material placed in the drying tower in the sixth step into a tubular furnace, introducing H2, calcining at the high temperature of 840-860 ℃ for 1.5-2.5H to obtain a FeP and CNT composite, cooling, taking out the FeP and CNT composite, and placing the FeP and CNT composite into the drying tower for later use;

step eight: weighing 40-60mg of the FeP and CNT compound placed in the drying tower in the seventh step, and grinding the FeP and CNT compound with 90-110mg of ionic surfactant for later use;

step nine: weighing 90-110mg of ionic surfactant, dissolving in deionized water, stirring uniformly, slowly adding the sample ground in the step eight, carrying out ultrasonic treatment for 0.5-1.5h, standing for 25-35min, removing large precipitates at the bottom, and taking the upper black dispersion liquid for later use;

step ten: dissolving 8-12ml of prepared GO liquid in 380-400ml of deionized water, and performing ultrasonic dispersion for 25-35min;

step eleven: slowly conveying the upper-layer black dispersion liquid CNT dispersion liquid prepared in the ninth step into the GO liquid prepared in the tenth step through a constant flow pump, and carrying out electrostatic self-assembly for 1.5-2.5 hours;

step twelve: weighing 450-550mg of ammonium bicarbonate, dissolving the ammonium bicarbonate in 8-12ml of deionized water, slowly adding the ammonium bicarbonate into the mixed solution obtained after the electrostatic self-assembly in the step eleven, carrying out electrostatic self-assembly for 25-35min again, carrying out suction filtration on the obtained solution to obtain a black precipitate FeP, CNT and GO compound, washing the compound for 2-4 times by using deionized water and absolute ethyl alcohol, drying and placing the compound into a drying tower for later use.

Preferred for the present invention are: the weight percentage of the phosphoric acid is 18-22%.

Preferred for the present invention are: the ionic surfactant is 3-sulfopropyl tetradecyl dimethyl betaine.

The invention has the beneficial effects that:

1. the enzyme activity of the nano-enzyme can be enhanced under the mild stimulation condition, and the nano-enzyme and the stimulation condition have synergistic effect to achieve the remarkable anti-tumor effect;

2. FePOs, CNTs, GO nano-enzyme complexes or FeP, CNT, GO nano-enzyme complexes possessing multiple enzyme activities and capable of multi-stimulus response can be prepared;

3. the enzyme activity is enhanced under the stimulation response, the tumor cells are killed, and a new idea is provided for tumor treatment by combining the stimulation response with the strategy of enzyme catalytic reaction.

Description of the drawings:

FIG. 1 is a graph of magnetic properties of FePOs, CNTs, GO nanoenzyme complexes or FeP, CNT, GO nanoenzyme complexes prepared by a preparation method according to an embodiment of the present invention;

fig. 2 is a schematic diagram of magnetic properties of FePOs, CNTs, and GO nanoenzyme complexes or FeP, CNTs, and GO nanoenzyme complexes prepared by a preparation method according to an embodiment of the present invention;

fig. 3 is a graph showing photo-thermal properties of FePOs, CNTs, and GO nanoenzyme complexes or FeP, CNTs, and GO nanoenzyme complexes prepared by a preparation method according to an embodiment of the present invention;

FIG. 4 is a schematic representation of peroxidase activity of FePOs, CNTs, GO nanoenzyme complexes or FeP, CNT, GO nanoenzyme complexes prepared by a preparation method according to an embodiment of the present invention;

fig. 5 is a table diagram of antitumor properties of FePOs, CNTs, GO nanoenzyme complexes or FeP, CNTs, GO nanoenzyme complexes prepared by the preparation method according to the embodiment of the present invention under different stimuli.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1:

a preparation method of a nano enzyme complex for treating tumors, wherein the nano enzyme complex is a complex of FePOs, CNTs and GO or a complex of FeP, CNTs and GO, and comprises the following steps:

the method comprises the following steps: putting CNT and steel ball into a ball milling tank according to a mass ratio of 45;

step two: taking 45mgCNT subjected to ball milling and drying in the step one, and adding 45ml of deionized water for ultrasonic dispersion for 0.5h for later use;

step three: adding 2g of urea, 0.2g of sodium dodecyl sulfate and 32ml of deionized water into a beaker in sequence, fully dissolving, adding the 40CNT solution dispersed in the step two, and uniformly stirring;

step four: weighing 0.05g of ferric sulfate, dissolving in 6ml of deionized water, slowly adding dropwise into the uniformly stirred solution in the step three, and continuously stirring for 15min;

step five: weighing 0.2g of phosphoric acid, dissolving in 6ml of deionized water, slowly dropwise adding the solution uniformly stirred in the step four, and continuously stirring for 15min;

step six: putting the mixed solution uniformly stirred in the step five into a reaction kettle, heating to 135 ℃, carrying out hydrothermal reaction for 1.5h, cooling to room temperature, carrying out suction filtration, washing for 2 times by using deionized water and absolute ethyl alcohol to obtain a FePOs and CNT compound, drying at 55 ℃, and putting into a drying tower for later use;

step seven: placing the FePOs and CNT composite material placed in the drying tower in the sixth step into a tubular furnace, introducing H2, calcining at 840 ℃ for 1.5H to obtain a FeP and CNT composite, cooling, taking out the FeP and CNT composite material, and placing the FeP and CNT composite material in the drying tower for later use;

step eight: weighing 40mg of the FeP and CNT compound placed in the drying tower in the seventh step, and grinding the FeP and CNT compound with 90mg of ionic surfactant for later use;

step nine: weighing 90mg of ionic surfactant, dissolving in deionized water, stirring uniformly, slowly adding the sample ground in the step eight, carrying out ultrasonic treatment for 0.5h, standing for 25min, removing large sediment at the bottom, and taking the upper black dispersion liquid for later use;

step ten: taking 8ml of prepared GO liquid, dissolving in 380ml of deionized water, and performing ultrasonic dispersion for 25min;

step eleven: slowly conveying the upper-layer black dispersion liquid CNT dispersion liquid prepared in the ninth step into the GO liquid prepared in the tenth step through a constant flow pump, and carrying out electrostatic self-assembly for 1.5h;

step twelve: weighing 450mg of ammonium bicarbonate, dissolving the ammonium bicarbonate in 8ml of deionized water, slowly adding the ammonium bicarbonate into the mixed solution obtained after the electrostatic self-assembly in the step eleven, carrying out electrostatic self-assembly for 25min again, carrying out suction filtration on the obtained solution to obtain a black precipitate FeP, CNT and GO compound, washing the compound for 2 times by using deionized water and absolute ethyl alcohol, drying the compound, and putting the compound into a drying tower for later use.

In the above examples, the phosphoric acid was 18% by weight.

In the above examples, the ionic surfactant was 3-sulfopropyltetradecyldimethyl betaine.

Example 2:

a preparation method of a nano enzyme compound for treating tumors, wherein the nano enzyme compound is a compound of FePOs, CNTs and GO or a compound of FeP, CNTs and GO, and comprises the following steps:

the method comprises the following steps: putting CNT and steel ball into a ball milling tank according to the mass ratio of 50;

step two: taking 50mgCNT subjected to ball milling and drying in the step one, and adding 50ml of deionized water for ultrasonic dispersion for 1h for later use;

step three: adding 3g of urea, 0.25g of sodium dodecyl sulfate and 34ml of deionized water into a beaker in sequence, fully dissolving, adding 50ml of CNT solution dispersed in the step two, and uniformly stirring;

step four: weighing 0.1g of ferric sulfate, dissolving in 8ml of deionized water, slowly adding into the uniformly stirred solution obtained in the third step, and continuously stirring for 20min;

step five: weighing 0.25g of phosphoric acid, dissolving in 8ml of deionized water, slowly dropwise adding the solution uniformly stirred in the step four, and continuously stirring for 20min;

step six: putting the mixed solution uniformly stirred in the fifth step into a reaction kettle, heating to 140 ℃, carrying out hydrothermal reaction for 2 hours, cooling to room temperature, carrying out suction filtration, washing for 3 times by using deionized water and absolute ethyl alcohol to obtain a FePOs and CNT compound, drying at 60 ℃, and putting into a drying tower for later use;

step seven: placing the FePOs and CNT composite material placed in the drying tower in the sixth step into a tubular furnace, introducing H2, calcining at 850 ℃ for 2H to obtain a FeP and CNT composite, cooling, taking out the FeP and CNT composite material, and placing the FeP and CNT composite material in the drying tower for later use;

step eight: weighing 50mg of the FeP/CNT composite placed in the drying tower in the seventh step, and grinding the FeP/CNT composite with 100mg of ionic surfactant for later use;

step nine: weighing 100mg of ionic surfactant, dissolving the ionic surfactant in deionized water, stirring uniformly, slowly adding the sample ground in the step eight, carrying out ultrasonic treatment for 1h, standing for 30min, removing large precipitates at the bottom, and taking the upper black dispersion liquid for later use;

step ten: dissolving 10ml of prepared GO liquid in 390ml of deionized water, and performing ultrasonic dispersion for 30min;

step eleven: slowly conveying the upper-layer black dispersion liquid CNT dispersion liquid prepared in the ninth step into the GO liquid prepared in the tenth step through a constant flow pump, and carrying out electrostatic self-assembly for 2 hours;

step twelve: weighing 500mg of ammonium bicarbonate, dissolving the ammonium bicarbonate in 10ml of deionized water, slowly adding the ammonium bicarbonate into the mixed solution obtained after the electrostatic self-assembly in the step eleven, carrying out electrostatic self-assembly for 30min again, carrying out suction filtration on the obtained solution to obtain a black precipitate FeP, CNT and GO compound, washing the compound for 3 times by using deionized water and absolute ethyl alcohol, drying the compound, and putting the compound into a drying tower for later use.

In the above examples, the phosphoric acid was 20% by weight.

Example 3:

the method comprises the following steps: putting CNT and steel ball into a ball milling tank according to the mass ratio of 55;

step two: adding 55ml of deionized water into the 55mgCNT subjected to ball milling and drying in the step one to perform ultrasonic dispersion for 1.5 hours for later use;

step three: adding 4g of urea, 0.3g of sodium dodecyl sulfate and 36ml of deionized water into a beaker in sequence, fully dissolving, adding 60ml of CNT solution dispersed in the step two, and uniformly stirring;

step four: weighing 0.15g of ferric sulfate, dissolving in 10ml of deionized water, slowly adding into the uniformly stirred solution obtained in the third step, and continuously stirring for 25min;

step five: weighing 0.3g of phosphoric acid, dissolving in 10ml of deionized water, slowly dropwise adding the solution uniformly stirred in the step four, and continuously stirring for 25min;

step six: putting the mixed solution uniformly stirred in the step five into a reaction kettle, heating to 145 ℃, carrying out hydrothermal reaction for 2.5h, cooling to room temperature, carrying out suction filtration, washing for 4 times by using deionized water and absolute ethyl alcohol to obtain a FePOs and CNT compound, drying at 65 ℃, and putting into a drying tower for later use;

step seven: placing the FePOs and CNT composite material placed in the drying tower in the sixth step into a tubular furnace, introducing H2, calcining at 860 ℃ for 2.5 hours to obtain a FeP and CNT composite, cooling, taking out the FeP and CNT composite material, and placing the FeP and CNT composite material in the drying tower for later use;

step eight: weighing 60mg of the FeP/CNT composite placed in the drying tower in the seventh step, and grinding the FeP/CNT composite with 110mg of ionic surfactant for later use;

step nine: weighing 110mg of ionic surfactant, dissolving the ionic surfactant in deionized water, stirring uniformly, slowly adding the sample ground in the step eight, carrying out ultrasonic treatment for 1.5h, standing for 35min, removing large sediment at the bottom, and taking the upper black dispersion liquid for later use;

step ten: dissolving 12ml of prepared GO liquid in 400ml of deionized water, and performing ultrasonic dispersion for 35min;

step eleven: slowly conveying the upper-layer black dispersion liquid CNT dispersion liquid prepared in the ninth step into the GO liquid prepared in the tenth step through a constant flow pump, and carrying out electrostatic self-assembly for 2.5 hours;

step twelve: weighing 550mg of ammonium bicarbonate, dissolving the ammonium bicarbonate in 12ml of deionized water, slowly adding the ammonium bicarbonate into the mixed solution obtained after the electrostatic self-assembly in the step eleven, carrying out electrostatic self-assembly for 35min again, carrying out suction filtration on the obtained solution to obtain a black precipitate FeP, CNT and GO compound, washing the black precipitate FeP, CNT and GO for 4 times by using deionized water and absolute ethyl alcohol, drying and placing the black precipitate FeP, CNT and GO into a drying tower for later use.

In the above examples, the phosphoric acid was 22% by weight.

Fig. 1 and 2 show magnetic performance diagrams of FePOs, CNTs, and GO nanoenzyme complexes or FeP, CNTs, and GO nanoenzyme complexes prepared by the preparation method according to the embodiment of the present invention.

As shown in the following table, the following table shows the conductivities of the FePOs, CNTs, GO nanoenzyme complexes or the FeP, CNTs, GO nanoenzyme complexes prepared by the preparation method described in the embodiment of the present invention;

as shown in fig. 3, fig. 3 is a photo-thermal performance diagram of FePOs, CNTs, GO nanoenzyme complexes or FeP, CNTs, GO nanoenzyme complexes prepared by the preparation method according to the embodiment of the present invention.

As shown in fig. 4, fig. 4 is a graph of peroxidase activity of FePOs, CNTs, GO nanoenzyme complexes or FeP, CNTs, GO nanoenzyme complexes prepared by the preparation method according to the embodiment of the present invention.

As shown in fig. 5, fig. 5 is a graph of antitumor performance of FePOs, CNTs, GO nanoenzyme complexes or FeP, CNTs, GO nanoenzyme complexes prepared by the preparation method according to the embodiment of the present invention under different stimuli.

The preparation method of the nanoenzyme complex for treating tumor described in the above embodiment can prepare FePOs, CNTs, GO nanoenzyme complex or FeP, CNTs, GO nanoenzyme complex having multiple enzyme activities and multi-stimulus response, having peroxidase activity, catalase activity and superoxide dismutase activity, simultaneously being capable of responding to magnetic, electric and optical stimuli, realizing that the nanoenzyme activity is enhanced under mild stimulus conditions, and simultaneously achieving a significant anti-tumor effect by synergistic action with the stimulus conditions, and exploring the mechanism of the nanoenzyme in the cytomology of anti-tumor;

the prepared FePOs, CNTs and GO nano-enzyme compound or FeP, CNT and GO nano-enzyme compound can realize multiple stimulation responses, the enzyme activity is enhanced under the stimulation response, the compound has a killing effect on tumor cells, and a new idea is provided for tumor treatment by combining the stimulation responses with a strategy of enzyme catalytic reaction;

the prepared FePOs, CNT, GO nano-enzyme compound or FeP, CNT, GO nano-enzyme compound can play a role in anti-tumor activity without the action of drugs, exogenous stimulation (electricity, magnetism, light, heat and the like) can cooperate with nano-enzyme to resist tumors under the condition of applying exogenous stimulation, and endogenous high H of the tumors is generated ₂ O ₂ The expression of (a) is coordinated with the anti-tumor treatment by the combination of peroxidase activity and exogenous stimulation, and the continuous action of the next stage is carried out after one stage of tumor treatment by the SOD enzyme activity and the catalase activity.

The above embodiments only express specific embodiments of the present invention, and the description is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. A preparation method of a nano-enzyme compound for treating tumors is disclosed, wherein the nano-enzyme compound is a compound of FePOs, CNTs and GO or a compound of FeP, CNTs and GO, and is characterized by comprising the following steps:

step three: adding 2-4g of urea, 0.2-0.3g of sodium dodecyl sulfate and 32-36ml of deionized water into a beaker in sequence, fully dissolving, adding 40-60ml of CNT solution dispersed in the step two, and uniformly stirring;

step six: putting the mixed solution uniformly stirred in the fifth step into a reaction kettle, heating to 135-145 ℃, carrying out hydrothermal reaction for 1.5-2.5h, cooling to room temperature, carrying out suction filtration, washing with deionized water and absolute ethyl alcohol for 2-4 times to obtain a FePOs and CNT compound, drying at 55-65 ℃, and putting into a drying tower for later use;

step seven: placing the FePOs and CNT composite material placed in the drying tower in the sixth step into a tubular furnace, introducing H2, calcining at the high temperature of 840-860 ℃ for 1.5-2.5H to obtain a FeP and CNT composite, cooling, taking out the FeP and CNT composite, and placing the FeP and CNT composite in the drying tower for later use;

step eight: weighing 40-60mg of the FeP/CNT composite placed in the drying tower in the seventh step, and grinding the FeP/CNT composite with 90-110mg of ionic surfactant for later use;

step eleven: slowly conveying the upper-layer black dispersion liquid CNT dispersion liquid prepared in the ninth step into the GO liquid prepared in the tenth step through a constant flow pump, and carrying out electrostatic self-assembly for 1.5-2.5h;

2. The method as claimed in claim 1, wherein the phosphoric acid is 18-22 wt%.

3. The method for preparing the nanoenzyme complex for use in the treatment of tumor according to claim 1 or 2, wherein the ionic surfactant is 3-sulfopropyltetradecyldimethyl betaine.