CN114621924A - Porous carbon sphere nanoenzyme-doped hydrogen bond organic framework shell layer and preparation method thereof - Google Patents
Porous carbon sphere nanoenzyme-doped hydrogen bond organic framework shell layer and preparation method thereof Download PDFInfo
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Abstract
The invention provides a hydrogen bond organic framework shell layer doped with porous carbon sphere nanoenzyme and a preparation method thereof, belonging to the technical field of organic framework shell layer preparation. The organic frame shell layer is formed by taking an organic construction unit as a frame main body material, uniformly doping porous carbon sphere nanoenzyme inside the organic frame shell layer, and directly growing on the surface of a cell through electrostatic interaction and hydrogen bond interaction; the organic building units are 1, 4-benzamidine hydrochloride and tetra (4-carboxyphenyl) methane. The rigidity of the organic framework shell layer and the shielding capacity to cells can ensure that the wrapped neural stem cells maintain totipotency, prevent cell membrane damage in the microinjection process, protect the cells to improve the survival rate of cell transplantation, and provide protection for the wrapped neural stem cells.
Description
Technical Field
The invention belongs to the technical field of organic framework shell layer preparation, and particularly relates to a porous carbon sphere nanoenzyme-doped hydrogen bond organic framework shell layer for assisting storage and implantation of neural stem cells and a preparation method thereof.
Background
Neural stem cells are a self-renewing cell population that can be isolated, proliferated, genetically manipulated and differentiated in vitro to produce neurons and glial cells of the developing brain for the compensation or renewal of neural networks for the development of synaptic connections to maintain normal neural function. Excellent totipotency, sufficient neural stem cell numbers, and a healthy transplantation microenvironment are key to successful stem cell transplantation. However, studies show that under in vitro culture conditions, neural stem cells inevitably undergo autodifferentiation due to cell-matrix interactions and the presence of cytokines, resulting in totipotency loss. In addition, during microinjection, external forces can damage cell membranes, resulting in reduced cell viability. Poor tissue microenvironments at the site of transplantation, such as high levels of oxidative stress, can also lead to reduced activity of transplanted neural stem cells. These several factors significantly limit the therapeutic efficacy of neural stem cell transplantation.
Therefore, the problems and challenges in the field of stem cell regeneration medicine are that totipotency loss is faced during the storage process of neural stem cells and the survival rate is faced low during the transplantation process.
Disclosure of Invention
The protective shell layer obviously reduces totipotency loss of neural stem cells in an in-vitro culture process, improves the cell survival rate in a cell transplantation process, and promotes the effect of a stem cell therapy.
The invention firstly provides a hydrogen bond organic frame shell layer doped with porous carbon sphere nanoenzyme, wherein the organic frame shell layer is formed by directly growing on the surface of a cell through electrostatic interaction and hydrogen bond interaction, wherein an organic construction unit is used as a frame main body material, and the porous carbon sphere nanoenzyme is uniformly doped in the organic frame shell layer;
the organic building units are 1, 4-benzamidine hydrochloride and tetra (4-carboxyphenyl) methane.
Preferably, the doping proportion of the porous carbon sphere nanoenzyme is 20%.
The invention also provides a preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme, which comprises the following steps:
the method comprises the following steps: dissolving a porous carbon sphere and 1, 4-benzamidine hydrochloride in a buffer solution, and uniformly mixing to obtain a solution A;
step two: adding monodisperse neural stem cells into the solution A obtained in the step one, removing a supernatant through shaking and centrifugation, and continuously adding a buffer solution into the precipitate to obtain a solution B;
step three: dissolving porous carbon spheres and tetra (4-carboxyphenyl) methane in a buffer solution, and uniformly mixing to obtain a solution C;
step four: and mixing the solution B and the solution C, and shaking and centrifuging to remove the supernatant to obtain the hydrogen bond organic frame layer doped with the porous carbon sphere nanoenzyme wrapped on the neural stem cells.
Preferably, the mass ratio of the porous carbon spheres to the 1, 4-benzenedicarboxamidine hydrochloride in the step one is 0.59: 0.78.
Preferably, the buffer of step one is phosphate buffer, pH 7.4.
Preferably, the mixing time of the first step is 5-10 min.
Preferably, the mass ratio of the porous carbon spheres in the step three to the tetra (4-carboxyphenyl) methane is 0.59: 1.56.
Preferably, the buffer solution of step three is phosphate buffer solution, pH 7.4.
Preferably, the mixing time of the third step is 5-10 min.
Preferably, the preparation method of the porous carbon sphere comprises the following steps:
1) mixing melamine, formalin solution and distilled water in a three-neck flask, stirring at 80 ℃ to obtain colorless transparent solution,
2) adding phenol, formalin aqueous solution and sodium hydroxide into the colorless and transparent solution, and stirring at 60 ℃ for 30min to obtain a mixed solution;
3) adding Pluronic F127 aqueous solution into the mixed solution, and stirring for 2 hours at 66 ℃ to obtain a product;
4) adding the product obtained in the third step into water to dilute the solution, after 20h, heating the solution in an autoclave at 130 ℃ for 24h, finally, centrifugally collecting the product, washing with water for several times, drying at room temperature, and drying the dried powder in N2Carbonizing at 800 ℃ for 3h in the atmosphere to obtain the porous carbon spheres.
The invention has the advantages of
The invention provides a hydrogen bond organic framework shell layer doped with porous carbon sphere nanoenzyme and a preparation method thereof. Firstly, the rigidity and the shielding capacity of the hydrogen bond organic framework to cells can ensure that the wrapped neural stem cells maintain totipotency; secondly, the rigid hydrogen-bonded organic framework prevents cell membrane damage in the microinjection process and protects cells to improve the survival rate of cell transplantation; third, the antioxidant activity of the porous carbon spheres mimics the enzyme activity, and provides protection to the encapsulated neural stem cells by neutralizing reactive oxygen radicals (superoxide anions, hydroxyl radicals) during storage and transplantation of the neural stem cells. Meanwhile, the following advantages are provided:
(1) the hydrogen bond organic framework shell layer has good biocompatibility, mild and simple preparation conditions, can greatly reduce the damage to cells, and has the advantages of convenient use, simple preparation, simple storage and the like.
(2) The protective shell layer can respond to the near-infrared light in the second region to degrade, releases wrapped cells, and is suitable for non-invasive control and release of cells in a living body.
Drawings
Fig. 1 is a graph showing the results of optimizing the doping ratio of porous carbon spheres in example 1 of the present invention.
FIG. 2 is a scanning electron micrograph and an X-ray powder diffraction pattern of the neural stem cell encapsulating process in example 2 of the present invention.
FIG. 3 is a scanning electron micrograph and an X-ray powder diffraction pattern of a neural stem cell releasing process in example 3 of the present invention.
Fig. 4 is a graph illustrating the effect of hydrogen bond organic framework doped with porous carbon spheres on protecting neural stem cells from oxidative stress damage in example 4 of the present invention.
FIG. 5 is a graph showing the effect of the hydrogen bonding organic framework doped with porous carbon spheres on reducing the damage of the neural stem cell membrane in example 5 of the present invention.
Fig. 6 is a graph of the study on the effect of the porous carbon sphere doped hydrogen bond organic framework on maintaining totipotency of neural stem cells in embodiment 6 of the present invention.
FIG. 7 is a graph of the effect of the hydrogen bond organic framework protective shell doped with porous carbon spheres on the recovery of the damaged neural network of the Alzheimer's disease model mouse in example 7 of the present invention.
FIG. 8 is a mechanism exploration diagram of a hydrogen bond organic framework protective shell doped with porous carbon spheres in recovering an impaired neural network of a mouse model of Alzheimer's disease in example 8 of the present invention.
Detailed Description
The invention firstly provides a hydrogen bond organic frame shell layer doped with porous carbon sphere nanoenzyme, wherein the organic frame shell layer is formed by directly growing on the surface of a cell through electrostatic interaction and hydrogen bond interaction, wherein an organic construction unit is used as a frame main body material, and the porous carbon sphere nanoenzyme is uniformly doped in the organic frame shell layer;
the organic building units are 1, 4-benzamidine hydrochloride and tetra (4-carboxyphenyl) methane.
The invention also provides a preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme, which comprises the following steps:
the method comprises the following steps: dissolving a porous carbon sphere and 1, 4-benzamidine hydrochloride in a buffer solution, and uniformly mixing to obtain a solution A; the mass ratio of the porous carbon spheres to the 1, 4-benzamidine hydrochloride is preferably 0.59:0.78,
the buffer solution is preferably phosphate buffer solution, pH is 7.4, and the mixing time is preferably 5-10 min;
step two: adding monodisperse neural stem cells (2X 10) into the solution A obtained in the step one∧6Cell separation), removing supernatant through shaking and centrifugation, and continuously adding a buffer solution into the precipitate to obtain a solution B; the shaking time is preferably 5-10min, and the centrifugation speed is preferably 2,000 Xg, 3 min; the buffer solution is preferably phosphate buffer solution, pH 7.4;
step three: dissolving porous carbon spheres and tetra (4-carboxyphenyl) methane in a buffer solution, and uniformly mixing to obtain a solution C; the mass ratio of the porous carbon spheres to the tetra (4-carboxyphenyl) methane is preferably 0.59:1.56,
the buffer solution is preferably phosphate buffer solution, pH is 7.4, and the mixing time is preferably 5-10 min;
step four: and mixing the solution B and the solution C, and shaking and centrifuging to remove the supernatant to obtain the hydrogen bond organic frame layer doped with the porous carbon sphere nanoenzyme wrapped on the neural stem cells. The shaking time is preferably 5-10min, and the centrifugation speed is preferably 2,000 Xg, 3 min.
According to the present invention, the neural stem cell is an isolated neural stem cell obtained by a culturing method conventionally used in the art, and is not particularly limited.
According to the present invention, the porous carbon spheres are prepared by a conventional preparation method in the art, and are not particularly limited, and preferably prepared by the following method:
1) mixing melamine, formalin aqueous solution and distilled water in a three-neck flask, and stirring at 80 ℃ to obtain colorless and transparent solution, wherein the mass g of the melamine is as follows: the volume mL of the formalin aqueous solution is preferably 1.798: 2.1; the concentration of the formalin aqueous solution is preferably 37.0 wt%;
2) adding phenol, formalin aqueous solution and sodium hydroxide into the colorless and transparent solution, and stirring at 60 ℃ for 30min to obtain a mixed solution; the mass g of the phenol is as follows: volume mL of formalin aqueous solution: the volume mL of sodium hydroxide is preferably 0.6: 2.1: 15; the concentration of the formalin aqueous solution is preferably 37.0 wt%; the concentration of sodium hydroxide is preferably 0.1 mol.L-1；
3) Adding Pluronic F127 aqueous solution into the mixed solution, and stirring at 66 ℃ for 2h to obtain a product, wherein the volume of the sodium hydroxide and Pluronic F127(0.7g) aqueous solution is preferably 1: 1; the stirring speed is preferably 350 r.min-1；
4) Adding the product of step three into water to dilute solution, after 20h, taking the solution and heating in an autoclave at 130 ℃ for 24h, finally, centrifuging and collecting the product, washing with water for several times and drying at room temperature, and drying the dried powder in N2Carbonizing at 800 deg.C for 3h in atmosphere to obtain porous carbon spheres with diameter of about 100nm, and storing at room temperature. The centrifugation speed is 12,000 Xg, 15 min.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 examination of organic framework shells (cell-free template) prepared with different doping ratios of porous carbon spheres
(1) 1.798g of melamine, 2.1mL of an aqueous formalin solution (37.0 wt%), and 15mL of distilled water were mixed in a three-necked flask and stirred at 80 ℃ to obtain a colorless transparent solution. Subsequently, 0.6g of phenol, 2.1mL of an aqueous formalin solution (37.0 wt%) and 15mL of sodium hydroxide (0.1 mol. multidot.L) were added thereto-1) Stirring at 60 deg.C for 30 min. 15mL of an aqueous solution of Pluronic F127(0.7g) were then added thereto at 350 r.min-1Stirring speed, stirring at 66 ℃ for 2 h. Next, 50mL of water was added to dilute the solution, and after 20h, 17.7mL of the solution was heated in an autoclave at 130 ℃ for 24 h. Finally, the product was collected by centrifugation (12,000 Xg, 15min), washed several times with water and dried at room temperature. Mixing the dried powder with N2Carbonizing at 800 deg.C for 3h in atmosphere to obtain porous carbon spheres with diameter of about 100nm, and storing at room temperature.
(2) Porous carbon spheres (0mg, 0.27mg, 0.59mg and 1.00mg) with different masses, 1, 4-benzamidine hydrochloride 0.78mg and tetra (4-carboxyphenyl) methane 1.56mg are mixed and stirred at room temperature to obtain the hydrogen bond organic framework doped with the porous carbon sphere nanoenzyme. Centrifugally washing and drying to obtain hydrogen bond organic framework materials (the doping contents are 0%, 10%, 20% and 30% wt% respectively), and suspending the solid in deionized water to obtain the final concentration of 1 mg/mL-1。
Then, samples with different proportions are respectively irradiated by 1064nm laser with the laser power of 0.8W cm-2The irradiation time is 0min, 5min and 10min respectively, and the temperature change of the solution in the whole process is recorded. And respectively taking samples in different illumination periods to prepare scanning samples, and recording the shape change. As shown in fig. 1, it can be seen that the dissociation of the hydrogen bonding organic framework is affected by the ratio of the doped porous carbon spheres,when the doping proportion is 20%, the hydrogen bond organic framework can be completely decomposed in 10min under illumination, and the solution temperature is 40 ℃. Therefore, the doping proportion of 20 percent is used as a parameter for preparing the final hydrogen bond organic framework doped by the porous carbon sphere nanoenzyme, and the laser power is fixed to be 0.8W-cm-2。
Example 2 preparation of porous carbon sphere nanoenzyme-doped Hydrogen bond organic framework layer wrapped on neural Stem cells
(1) 1.798g of melamine, 2.1mL of an aqueous formalin solution (37.0 wt%), and 15mL of distilled water were mixed in a three-necked flask and stirred at 80 ℃ to obtain a colorless transparent solution. Subsequently, 0.6g of phenol, 2.1mL of an aqueous formalin solution (37.0 wt%) and 15mL of sodium hydroxide (0.1 mol. multidot.L) were added thereto-1) Stirring at 60 deg.C for 30 min. 15mL of an aqueous solution of Pluronic F127(0.7g) were then added thereto at 350 r.min-1Stirring speed, stirring at 66 ℃ for 2 h. Next, 50mL of water was added to dilute the solution, and after 20h, 17.7mL of the solution was heated in an autoclave at 130 ℃ for 24 h. Finally, the product was collected by centrifugation (12,000 Xg, 15min), washed several times with water and dried at room temperature. Mixing the dried powder with N2Carbonizing at 800 deg.C for 3h in atmosphere to obtain porous carbon spheres with diameter of about 100nm, and storing at room temperature.
(2) Dissolving the porous carbon spheres (0.59mg) obtained in the step (1) and 1, 4-benzamidine hydrochloride (0.78mg) in a phosphate buffer (pH 7.4, 1mL) and stirring for 10min until uniform mixing to obtain a solution A.
(3) Adding monodisperse neural stem cells (2X 10) into the solution A obtained in the step (2)∧6Individual cells), gently shaken at room temperature for 5min, centrifuged (2,000 Xg, 3min) to remove the supernatant, and phosphate buffer (pH 7.4, 1mL) was added to the pellet to obtain solution B.
(4) The porous carbon spheres (0.59mg) obtained in step (1) and tetrakis (4-carboxyphenyl) methane (1.56mg) were dissolved in phosphate buffer (pH 7.4, 1mL) and stirred for 10min until well mixed, yielding solution C.
(5) And (3) mixing the solution B and the solution C obtained in the steps (3) and (4), slightly shaking at room temperature for 10min, centrifuging (2,000 Xg, 3min) to remove supernatant to obtain the porous carbon sphere nanoenzyme-doped hydrogen bond organic framework layer wrapped on the neural stem cells, storing the porous carbon sphere nanoenzyme-doped hydrogen bond organic framework layer in a common culture medium, and periodically replacing the culture medium every two days.
Preparing a cell scanning sample by a gradual dehydration mode, and specifically comprising the following operation steps: gradually infiltrating the cells into a phosphate buffer solution containing 0%, 15%, 30%, 45%, 60%, 70%, 80%, 90% and 100% of ethanol, finally centrifugally collecting the cells, immersing the cells in a glutaraldehyde solution, and directly dripping the glutaraldehyde solution on a clean silicon chip. As shown in fig. 2, fig. 2a is an X-ray powder diffraction pattern of a hydrogen bond organic framework layer doped with porous carbon sphere nanoenzyme wrapped on a neural stem cell, fig. 2b is a scanning electron microscope photograph of an unwrapped cell and a wrapped cell under different multiplying powers, the scanning electron microscope structure shows that the cell surface has a hydrogen bond organic framework shell layer uniformly wrapped, and the hydrogen bond organic framework can be identified to be uniformly wrapped on the cell surface and have good crystallinity by combining with a characteristic peak of the hydrogen bond organic framework of the X-ray powder diffraction.
Example 3 neural Stem cell Release Process
The neural stem cells coated with the hydrogen bond organic framework prepared in example 2 were placed in a phosphate buffer (pH 7.4) and irradiated with a laser at 1064nm with a laser power of 0.8 W.cm-2An intermittent irradiation mode is adopted, namely 2min +3min +2min +3min, and the total irradiation time is 10 min. Cells were collected by centrifugation (2,000 Xg, 3min) and dehydrated to prepare a scanning sample. As shown in fig. 3, fig. 3a is a scanning electron micrograph of the degraded cell shell layer, fig. 3b is an X-ray powder diffraction pattern of the degraded cell shell layer, the scanning electron micrograph shows that illumination enables the hydrogen bond organic framework shell layer with rough cell surface to fall off, and the hydrogen bond organic framework can be determined to be dissociated after illumination and release the wrapped cells by combining the result of the X-ray powder diffraction.
Example 4 research on the Effect of porous carbon sphere-doped hydrogen bond organic frameworks on protecting neural stem cells from oxidative stress damage
The neural stem cells coated with the hydrogen-bonding organic framework prepared in example 2 were placed in phosphate buffer (pH 7.4, containing 0.1mM hydrogen peroxide) and cultured at 37 ℃ for 28 h. Then, after removing the shell layer by applying illumination (conditions are the same as above), the cell viability was examined immediately by using a CCK-8 kit, and the reactive oxygen species level in the cells was detected by using a reactive oxygen species probe. As shown in fig. 4, fig. 4a is a fluorescence image of the total level of active oxygen in cells, 4b is a flow cytometry analysis result of the total level of active oxygen in cells, and 4c is a cell viability analysis result of hydrogen bond organic framework shell layer resisting oxidation damage. The results show that the cells doped with hydrogen bond organic frameworks of the porous carbon sphere nanoenzyme have higher survival rate, and the intracellular reactive oxygen species level is lower than that of the control group. Therefore, the porous carbon sphere doped hydrogen bond organic framework has the effect of remarkably protecting the neural stem cells from oxidative stress damage.
Example 5 Effect of porous carbon sphere-doped Hydrogen bond organic frameworks on reducing neural Stem cell Membrane injury
The neural stem cells wrapped with the hydrogen bond organic framework prepared in the example 2 are placed in physiological saline, and the cell density is 2.5 multiplied by 107cells·mL-1. Then, the cell solution was aspirated by a 10uL Hamilton syringe with a 33-gauge needle at an injection rate of 1. mu.l.min-1The injection time is 4 min. Cells were injected into 24-well plates containing 0.5mL of medium. After 30min, cell viability was verified by a live-dead staining method and a CCK-8 analysis method, respectively, and damage to cell membranes was tested by pyridine iodide. As shown in fig. 5, fig. 5a is a staining diagram of cell membrane damage, and fig. 5b is a proportion diagram of cell membrane damage, and the result shows that the neural stem cells wrapped by hydrogen bond organic frameworks have less cell membrane damage and higher cell viability during microinjection. This demonstrates that the hydrogen bonded organic framework of the present invention is able to resist membrane damage during microinjection, better preserving cell viability.
Example 6 research on the Effect of porous carbon sphere-doped Hydrogen bond organic frameworks on maintaining totipotency of neural Stem cells
The neural stem cells coated with the hydrogen bond organic framework prepared in example 2 were cultured in DMEM/F12 medium containing 1% fetal bovine serum without cytokines bFGF and EGF for 10 days, and then the shell layer was removed, and then Nestin immunofluorescence staining was performed on the cells to verify the dryness of the neural stem cells. The released neural stem cells were then cultured for a further 14 days and cell differentiation was verified by immunofluorescence staining (neurons Tuj 1, glial GFAP). As shown in FIG. 6, FIG. 6a is a photograph of the Nestin staining, FIG. 6b is a photograph of the flow analysis of the Nestin staining, FIG. 6c is a photograph of the formation of cytosphere, FIG. 6d is a photograph of the differentiation of cells, and the results of the photographs taken in combination with the formation of the neurosphere show that the encapsulated neural stem cells can maintain the dryness after 10 days of culture, and the differentiation behavior is normal after the cells are released. The totipotency of the neural stem cells can be effectively preserved by the hydrogen bond organic framework shell layer, and the cell differentiation behavior is not influenced.
Example 7 Effect study of porous carbon sphere-doped hydrogen bond organic framework protective shell layer on recovery of damaged neural network of mouse model of Alzheimer's disease
Establishing an Alzheimer's disease mouse model (APPswe/PS1M146V/TauP301L) and feeding the mouse model until 10 months of dementia, injecting the neural stem cells wrapped with hydrogen bond organic frameworks to the two sides of the brain of the mouse in a stereotactic way, wherein the injection volume is 5uL, and the injection speed is 1 uL.min-1The injection position is as follows: the fore chimney is taken as the origin of coordinates, and the fore position and the rear position are-1.70 mm; the middle side position is 1.50 mm; dorsal and ventral-1.50 mm. After injection, 1064nm light was applied to release the encapsulated cells. And 2 months later, the water maze test is carried out on the mouse to verify the recovery condition of the spatial memory ability. As shown in fig. 7, fig. 7a is escape latency, fig. 7b is target quadrant dwell time, fig. 7c is number of times of crossing target platform, fig. 7d is swimming speed, and fig. 7e is swim path diagram. The result shows that the mice in the neural stem cell treatment group which is wrapped by the hydrogen bond organic framework doped with the porous carbon sphere nanoenzyme are injected with the neural stem cell treatment group and have the advantages of higher swimming speed, shorter latency, more times of crossing a target platform and longer residence time in a target quadrant. The result shows that the hydrogen bond organic framework shell layer auxiliary neural stem cell transplantation doped with the porous carbon sphere nanoenzyme has the treatment effect of better recovering the memory disorder of the transgenic Alzheimer's disease model mouse.
Example 8 mechanism exploration of porous carbon sphere doped hydrogen bond organic framework protective shell in recovery of damaged neural network of alzheimer model mouse
And (3) taking out the brain tissue of the mouse subjected to the water maze experiment, carrying out Tuj 1 staining and Nie's staining, and detecting the lipid peroxidation level of the brain tissue and the total active oxygen level of the brain. As shown in fig. 8, fig. 8a is Tuj 1 immunofluorescent staining, fig. 8b is brain lipid peroxidation level, fig. 8c is brain total reactive oxygen species level fig. 8d is niemann's staining pattern. The results show that the number of the new neurons in the brain of the mice in the group treated by injecting the hydrogen bond organic framework coated by the porous carbon sphere nanoenzyme is increased, the health state of the neurons is better, and the lipid peroxidation level and the total active oxygen level are lower. The result shows that the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme provided by the invention assists the transplantation therapeutic effect of the neural stem cells from the synergistic effect of the shell layer on the physical protection of the neural stem cells and the reduction of the oxidation pressure of the transplantation site.
Claims (10)
1. A hydrogen bond organic frame shell layer doped with porous carbon sphere nanoenzyme is characterized in that the organic frame shell layer is formed by directly growing on the surface of a cell through electrostatic interaction and hydrogen bond interaction, wherein an organic construction unit is used as a frame main body material, and the porous carbon sphere nanoenzyme is uniformly doped in the organic frame shell layer;
the organic building units are 1, 4-benzamidine hydrochloride and tetra (4-carboxyphenyl) methane.
2. The hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme as claimed in claim 1, wherein the doping proportion of the porous carbon sphere nanoenzyme is 20%.
3. The preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme, which is characterized by comprising the following steps of:
the method comprises the following steps: dissolving a porous carbon sphere and 1, 4-benzamidine hydrochloride in a buffer solution, and uniformly mixing to obtain a solution A;
step two: adding monodisperse neural stem cells into the solution A obtained in the step one, removing a supernatant through shaking and centrifugation, and continuously adding a buffer solution into the precipitate to obtain a solution B;
step three: dissolving porous carbon spheres and tetra (4-carboxyphenyl) methane in a buffer solution, and uniformly mixing to obtain a solution C;
step four: and mixing the solution B and the solution C, and shaking and centrifuging to remove the supernatant to obtain the hydrogen bond organic frame layer doped with the porous carbon sphere nanoenzyme wrapped on the neural stem cells.
4. The method for preparing the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme according to claim 3, wherein the mass ratio of the porous carbon sphere in the first step to the 1, 4-benzenedicarboxamidine hydrochloride is 0.59: 0.78.
5. The preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme as claimed in claim 3, wherein the buffer solution in the first step is a phosphate buffer solution with pH of 7.4.
6. The preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme as claimed in claim 3, wherein the mixing time of the first step is 5-10 min.
7. The preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme as claimed in claim 3, wherein the mass ratio of the porous carbon sphere in the step three to the tetrakis (4-carboxyphenyl) methane is 0.59: 1.56.
8. The preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme as claimed in claim 3, wherein the buffer solution in the third step is a phosphate buffer solution with pH of 7.4.
9. The preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme as claimed in claim 3, wherein the mixing time of the third step is 5-10 min.
10. The preparation method of the hydrogen bond organic framework shell layer doped with the porous carbon sphere nanoenzyme as claimed in claim 3, wherein the preparation method of the porous carbon sphere comprises the following steps:
1) mixing melamine, formalin aqueous solution and distilled water in a three-neck flask, and stirring at 80 ℃ to obtain colorless and transparent solution;
2) adding phenol, formalin aqueous solution and sodium hydroxide into the colorless and transparent solution, and stirring at 60 ℃ for 30min to obtain a mixed solution;
3) adding Pluronic F127 aqueous solution into the mixed solution, and stirring for 2 hours at 66 ℃ to obtain a product;
4) adding the product of step three into water to dilute solution, after 20h, taking the solution and heating in an autoclave at 130 ℃ for 24h, finally, centrifuging and collecting the product, washing with water for several times and drying at room temperature, and drying the dried powder in N2Carbonizing at 800 ℃ for 3h in the atmosphere to obtain the porous carbon spheres.
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