CN112938956B - Magnetic graphene oxide capable of adsorbing beta amyloid protein and preparation method thereof - Google Patents

Abstract

The invention discloses magnetic graphene oxide capable of adsorbing beta amyloid and a preparation method thereof, wherein the magnetic graphene oxide comprises magnetic graphene oxide, a polyethyleneimine matrix, a silane coupling agent and folic acid, the polyethyleneimine matrix is synthesized by the silane coupling agent, the graphene oxide is compounded on the polyethyleneimine matrix, and the folic acid is loaded on the graphene oxide; the graphene oxide has a high specific surface area and rich surface functional groups, so that the graphene oxide has certain adsorption capacity, the graphene oxide magnetic material is subjected to graft modification by using polyethyleneimine, and a novel adsorption material obtained by compounding safe folic acid has a good adsorption effect on beta amyloid protein.

Description

Magnetic graphene oxide capable of adsorbing beta amyloid and preparation method thereof

technical field

The invention relates to the technical field of magnetic graphene oxide, in particular to a magnetic graphene oxide capable of adsorbing beta amyloid and a preparation method thereof.

Background technique

The molecular weight of β-amyloid is about 4kDa, which is derived from the proteolysis of β-amyloid precursor and secreted by cells. It has a strong neurotoxic effect after the accumulation of cell matrix. β-amyloid is produced from amyloid precursor protein and can be produced by a variety of cells, circulating in blood, cerebrospinal fluid and brain interstitial fluid, most of which are bound to chaperone molecules, and a few exist in free state. Among them, beta amyloid is associated with Alzheimer's disease and cerebral amyloid angiopathy. The process of β-amyloid-induced disease is: the excessive production of the neurotoxic substance β-amyloid (Aβ) in the brain leads to the accumulation of Aβ in the brain → degeneration and death of nerve cells → brain atrophy → dementia. Aβ deposition is one of the important pathological features of Alzheimer's disease (AD). A therapeutic strategy targeting Aβ clearance is one of the current mainstream research directions.

Graphene oxide has a high specific surface area and abundant surface functional groups, which makes it have a certain adsorption capacity, but the existing graphene oxide has a general adsorption effect on beta amyloid, and at the same time, because it does not have magnetism, it cannot be carried out. Magnetic recycling, poor practicality.

SUMMARY OF THE INVENTION

The object of the invention is to provide a magnetic graphene oxide capable of adsorbing beta amyloid and a preparation method thereof. The invention has the advantages of good adsorption effect on beta amyloid and relatively simple preparation, so as to solve the problems proposed in the above background technology. question.

To achieve the above object, the present invention provides the following technical solutions:

Magnetic graphene oxide that can adsorb beta amyloid, including magnetic graphene oxide, polyethyleneimine matrix, silane coupling agent and folic acid, the polyethyleneimine matrix is synthesized by silane coupling agent, and graphene oxide is compounded in On polyethyleneimine matrix, folic acid is supported on graphene oxide.

Another technical problem to be solved by the present invention is to provide a preparation method of magnetic graphene oxide that can adsorb beta amyloid, comprising the following steps:

S1: preparation of magnetic graphene oxide; 15-30 mg of graphene oxide is weighed and dissolved in 15-30 ml of diethylene glycol solution, stirred evenly, and then the mixed solution is put into ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing is controlled In 26-30min, then under the action of magnetic stirring, add sodium acetate 150-250mg, ferric chloride hexahydrate 100-200mg in turn to form a uniform yellow suspension, place the suspension in the reaction kettle, the The temperature was adjusted to 200°C, and the suspension was reacted in the reactor at 200°C for 8-10h, then the suspension was placed in a centrifuge, and the suspension was centrifuged at 3000r/min for 5min, and then the sediment The upper supernatant is poured out to obtain a precipitate, and the precipitate is washed 10-20 times with 100-400ml of absolute ethanol and 200-350ml of distilled water in turn to obtain a high-quality magnetic graphene oxide precipitate, which is then subjected to high-quality magnetic oxidation. The graphene precipitate is vacuum-dried to obtain magnetic graphene oxide;

S2: Preparation of polyethyleneimine matrix:

a Preparation of polyethyleneimine emulsion: take 8-10 mL of polyethyleneimine aqueous solution with a mass percentage of 20%, 10-12 mL of liquid paraffin and 2 mL of polyvinyl alcohol porogen to mix, and then add surfactant to the mixture A mixed solution is formed, and the final concentration of the surfactant is 0.04-0.06 g/mL, and then the mixed solution is put into an ultrasonic device for ultrasonic mixing, and the time of ultrasonic mixing is controlled at 2-3 min to form a polyethyleneimine emulsion;

b Pre-cross-linking polymerization reaction: take 15-20 mL of the above polyethyleneimine emulsion, then slowly add 15-20 mL of cross-linking agent dropwise under the action of magnetic stirring, and pre-cross-link for 2-3 hours;

c Cross-linking polymerization reaction: under the condition of magnetic stirring, take 20 mL of the pre-cross-linking mixture prepared by the above-mentioned pre-cross-linking polymerization reaction, slowly add 5-10 mL of sodium hydroxide solution dropwise, and control the concentration of the sodium hydroxide solution at 1.0-1. 2.0mol/L, after the reaction, the mixture is centrifuged, and the supernatant is removed by centrifugation to obtain a polyethyleneimine matrix;

S3: Preparation of polyethyleneimine-modified magnetic graphene oxide: Weigh 20-40 mg of magnetic graphene oxide and dissolve it in 20-40 ml of pH 7.4 phosphate buffer, and then put the mixture into ultrasonic equipment for ultrasonic mixing , the ultrasonic mixing time is controlled at 5-10min to make it disperse uniformly; then add carbodiimide hydrochloride 100-200mg, N-hydroxysuccinimide 50-117mg, polyethyleneimine to the mixed solution in turn The matrix is 200-400 mg, and after stirring evenly, it is filtered to obtain a filter cake. The filter cake is repeatedly washed 10 times with 400-1000 ml of ultrapure water to obtain a polyethyleneimine-modified magnetic graphene oxide precipitate. The amine-modified magnetic graphene oxide precipitate is vacuum-dried, the temperature of vacuum drying is controlled at 40-60 °C, and the drying time is 1-2 days, and finally polyethyleneimine-modified magnetic graphene oxide is obtained;

S4: Preparation of polyethyleneimine-modified magnetic graphene oxide loaded with folic acid: 5-20 mg of polyethyleneimine-modified magnetic graphene oxide was weighed and dissolved in 5-20 ml of ultrapure water, and then silane coupling agent 1- 4 mg of folic acid powder and 5-20 mg of folic acid powder, and then put the mixed solution into ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing is controlled within 1-2 hours to obtain the polyethyleneimine-modified magnetic graphene oxide loaded with folic acid.

Preferably, in the S2, the added quality of the crosslinking agent is the same as that of the polyethyleneimine emulsion.

Preferably, in the step S2, the obtained polyethyleneimine matrix needs to be washed 5-10 times with isopropanol, anhydrous ether, and distilled water, respectively.

Preferably, in the S4, the silane coupling agent is 3-aminopropyltriethoxysilane.

Compared with the prior art, the beneficial effects of the present invention are as follows: the magnetic graphene oxide capable of adsorbing beta amyloid and its preparation method proposed by the present invention comprise magnetic graphene oxide, polyethyleneimine matrix, silane coupling agent and folic acid, polyethyleneimine matrix is synthesized by silane coupling agent, graphene oxide is compounded on polyethyleneimine matrix, folic acid is loaded on graphene oxide, and the preparation method includes, preparation of magnetic graphene oxide, polyethyleneimine oxide Preparation of amine emulsion, preparation of polyethyleneimine-modified magnetic graphene oxide and preparation of folic acid-loaded polyethyleneimine-modified magnetic graphene oxide; wherein graphene oxide has a high specific surface area and abundant surface functional groups, making it It has a certain adsorption capacity. The graphene oxide magnetic material is grafted and modified by polyethyleneimine, and the new adsorption material obtained by compounding safe folic acid has a good adsorption effect on beta amyloid. It is injected into the cerebrospinal fluid. After the β-amyloid is adsorbed in the medium, it can be recovered by magnetic recovery, and the removal of β-amyloid can be realized. At the same time, the preparation method of magnetic graphene oxide is relatively simple and environmentally friendly.

Description of drawings

Fig. 1 is the overall process flow diagram of the present invention;

Fig. 2 is the process flow diagram of the preparation polyethyleneimine matrix of the present invention;

Fig. 3 is the experimental route of the mouse water maze experiment without the invention of the present invention;

Figure 4 is the experimental route of the invention using the mouse water maze of the invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

A magnetic graphene oxide capable of adsorbing beta amyloid, comprising magnetic graphene oxide, polyethyleneimine matrix, silane coupling agent and folic acid, polyethyleneimine matrix is synthesized by silane coupling agent, graphene oxide is compounded in On polyethyleneimine matrix, folic acid is supported on graphene oxide.

Example 1

Please refer to FIG. 1, a preparation method of magnetic graphene oxide that can adsorb beta amyloid, comprising the following steps:

The first step: preparation of magnetic graphene oxide; 25mg of graphene oxide was weighed and dissolved in 25ml of diethylene glycol solution, stirred evenly, and then the mixed solution was put into ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing was controlled at 30min , then under the action of magnetic stirring, add sodium acetate 150mg, ferric chloride hexahydrate 100mg successively to form a uniform yellow suspension, the suspension is placed in the reactor, the temperature in the reactor is adjusted to 200 ° C, and The suspension was reacted in the reactor at 200 °C for 8 h, then the suspension was placed in a centrifuge, the suspension was centrifuged at 3000 r/min for 5 min, and then the supernatant above the precipitate was poured out to obtain a precipitate , and the precipitate was washed 15 times with 400ml of absolute ethanol and 250ml of distilled water in turn to obtain a high-quality magnetic graphene oxide precipitate, and then the high-quality magnetic graphene oxide precipitate was vacuum-dried to obtain magnetic graphene oxide;

The second step: preparation of polyethyleneimine emulsion: mix 10 mL of polyethyleneimine aqueous solution with a mass percentage of 20%, 10 mL of liquid paraffin and 2 mL of polyvinyl alcohol porogen, and then add surfactant to the mixture to form Mixed liquid, the final concentration of surfactant is 0.04g/mL, then put the mixed liquid into ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing is controlled at 3min to form polyethyleneimine emulsion;

Step 3: Preparation of polyethyleneimine-modified magnetic graphene oxide: Weigh 40 mg of magnetic graphene oxide and dissolve it in 40 ml of pH 7.4 phosphate buffer, then put the mixture into an ultrasonic device for ultrasonic mixing. The mixing time is controlled at 5min to make it evenly dispersed; then 100mg of carbodiimide hydrochloride, 50mg of N-hydroxysuccinimide, and 200mg of polyethyleneimine emulsion are added to the mixed solution in turn, and the mixture is stirred evenly and used for Filtration to obtain a filter cake, and the filter cake was repeatedly washed 10 times with 1000 ml of ultrapure water to obtain a polyethyleneimine-modified magnetic graphene oxide precipitate, and then the polyethyleneimine-modified magnetic graphene oxide precipitate was vacuum-dried , the temperature of vacuum drying is controlled at 40-60 °C, and the drying time is 1 day, and finally polyethyleneimine-modified magnetic graphene oxide is obtained;

Step 4: Preparation of polyethyleneimine-modified magnetic graphene oxide loaded with folic acid: Weigh 20 mg of polyethyleneimine-modified magnetic graphene oxide and dissolve it in 20 ml of ultrapure water, then add 4 mg of silane coupling agent and folic acid powder 20mg, the silane coupling agent is 3-aminopropyltriethoxysilane, then the mixed solution is put into ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing is controlled within 2 hours to obtain folic acid-loaded polyethyleneimine modified of magnetic graphene oxide.

Embodiment 2

Please refer to Figure 1-2, a preparation method of magnetic graphene oxide that can adsorb beta amyloid, including the following steps:

The first step: preparation of magnetic graphene oxide; 30 mg of graphene oxide is weighed and dissolved in 30 ml of diethylene glycol solution, stirred evenly, and then the mixed solution is put into ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing is controlled at 30min , then under the action of magnetic stirring, add sodium acetate 250mg, ferric chloride hexahydrate 200mg successively to form a uniform yellow suspension, the suspension is placed in the reactor, the temperature in the reactor is adjusted to 200 ° C, and The suspension was reacted in the reactor at 200 °C for 10 hours, then the suspension was placed in a centrifuge, and the suspension was centrifuged at 3000 r/min for 5 minutes, and then the supernatant above the sediment was poured out to obtain a sediment , the precipitate was washed 20 times with 300ml of absolute ethanol and 350ml of distilled water in turn to obtain a high-quality magnetic graphene oxide precipitate, and then the high-quality magnetic graphene oxide precipitate was vacuum-dried to obtain magnetic graphene oxide;

The second step: the preparation of polyethyleneimine matrix:

a Preparation of polyethyleneimine emulsion: mix 10 mL of polyethyleneimine aqueous solution with a mass percentage of 20%, 10 mL of liquid paraffin and 2 mL of polyvinyl alcohol porogen, and then add surfactant to the mixed solution to form a mixed solution, The final concentration of the surfactant is 0.06g/mL, then the mixed solution is put into the ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing is controlled at 3min to form a polyethyleneimine emulsion;

b Pre-crosslinking polymerization reaction: take 20mL of the above polyethyleneimine emulsion, then slowly add 20mL of crosslinking agent dropwise under the action of magnetic stirring, and pre-crosslink for 2 hours. The added quality is the same;

c cross-linking polymerization reaction: under the condition of magnetic stirring, take 20 mL of the pre-cross-linking mixture prepared by the above-mentioned pre-cross-linking polymerization reaction, slowly add 10 mL of sodium hydroxide solution dropwise, and control the concentration of sodium hydroxide solution at 2.0 mol/L After the reaction, the mixture is centrifuged, and the supernatant is removed by centrifugation to obtain a polyethyleneimine matrix. The polyethyleneimine matrix needs to be washed 10 times with isopropanol, anhydrous ether and distilled water respectively.

Step 3: Preparation of polyethyleneimine-modified magnetic graphene oxide: Weigh 20 mg of magnetic graphene oxide and dissolve it in 20 ml of pH 7.4 phosphate buffer, then put the mixture into an ultrasonic device for ultrasonic mixing. The mixing time is controlled at 5min to make it evenly dispersed; then 100mg of carbodiimide hydrochloride, 100mg of N-hydroxysuccinimide, and 300mg of polyethyleneimine matrix are added to the mixed solution in turn, and the mixture is stirred evenly and used for Filtration to obtain a filter cake, and the filter cake was repeatedly washed 10 times with 1000 ml of ultrapure water to obtain a polyethyleneimine-modified magnetic graphene oxide precipitate, and then the polyethyleneimine-modified magnetic graphene oxide precipitate was vacuum-dried , the temperature of vacuum drying is controlled at 40-60 °C, and the drying time is 1 day, and finally polyethyleneimine-modified magnetic graphene oxide is obtained;

Step 4: Preparation of polyethyleneimine-modified magnetic graphene oxide loaded with folic acid: 20 mg of polyethyleneimine-modified magnetic graphene oxide was weighed and dissolved in 20 ml of ultrapure water, and then 3 mg of silane coupling agent and folic acid powder were added. 20mg, the silane coupling agent is 3-aminopropyltriethoxysilane, then the mixed solution is put into ultrasonic equipment for ultrasonic mixing, and the time of ultrasonic mixing is controlled within 2 hours to obtain folic acid-loaded polyethyleneimine modified of magnetic graphene oxide.

It is currently known that Aβ deposition is one of the important pathological features of Alzheimer's disease (AD), and is also a common pathway for AD induced by various causes, and is also a key factor in the formation and development of AD. A therapeutic strategy targeting Aβ clearance is one of the current mainstream research directions. The magnetic graphene oxide that can adsorb β-amyloid protein proposed by the present invention has broad application prospects in the research and development of β-amyloid-related diseases. Ideal for cleaning materials. In the behavioral experiment of the Alzheimer's disease mouse model, that is, the Morris water maze experiment, the comparison diagrams before and after the use of the present invention are shown in Figures 3 and 4. Figure 3 is the mouse path without the use of the present invention. Figure 4 shows the mouse route without the invention. It is confirmed by neurobehavioral experiments that the invention can effectively improve the cognitive ability of Alzheimer's disease model mice. The recyclable advantage can also play a role in beta amyloid detection and laboratory waste disposal, further broadening the application scenarios. The present invention provides a new idea for drug research and development of beta amyloid-related diseases including Alzheimer's disease, and is expected to become a preventive and therapeutic drug for Alzheimer's disease.

In summary, the magnetic graphene oxide capable of adsorbing beta amyloid and its preparation method proposed by the present invention include magnetic graphene oxide, polyethyleneimine matrix, silane coupling agent and folic acid, polyethyleneimine matrix Synthesized by silane coupling agent, graphene oxide is compounded on polyethyleneimine matrix, and folic acid is loaded on graphene oxide. The preparation method includes: preparation of magnetic graphene oxide, preparation of polyethyleneimine emulsion, preparation of polyethyleneimine Amine-modified magnetic graphene oxide and preparation of folic acid-loaded polyethyleneimine-modified magnetic graphene oxide; wherein graphene oxide has a high specific surface area and abundant surface functional groups, which make it have a certain adsorption capacity. Ethyleneimine has grafted modified graphene oxide magnetic material, and the new adsorption material obtained by compounding safe folic acid has good adsorption effect on beta amyloid, and it can be injected into cerebrospinal fluid to adsorb beta amyloid. Through magnetic recovery, the removal of beta amyloid can be achieved, and the preparation method of magnetic graphene oxide is relatively simple and environmentally friendly.

It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any relationship between these entities or operations. any such actual relationship or sequence exists. Moreover, the terms "comprising", "comprising" or any other variation thereof are intended to encompass non-exclusive inclusion such that a process, method, article or device comprising a list of elements includes not only those elements, but also includes not explicitly listed or other elements inherent to such a process, method, article or apparatus.

The above is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

Claims (5)

1. The preparation method of the magnetic graphene oxide capable of adsorbing the amyloid beta is characterized by comprising the following steps of:

s1: preparing magnetic graphene oxide; weighing 15-30mg of graphene oxide, dissolving the graphene oxide in 15-30ml of diethylene glycol solution, uniformly stirring, putting the mixed solution into ultrasonic equipment for ultrasonic mixing, controlling the ultrasonic mixing time to be 26-30min, then sequentially adding 250mg of sodium acetate and 200mg of ferric chloride hexahydrate under the action of magnetic stirring to form uniform yellow suspension, putting the suspension into a reaction kettle, adjusting the temperature level in the reaction kettle to 200 ℃, enabling the suspension to react in the reaction kettle for 8-10h at 200 ℃, then putting the suspension into a centrifuge, centrifuging the suspension for 5min at the revolution of 3000r/min, then pouring out the supernatant above the precipitate to obtain the precipitate, centrifuging and washing the precipitate for 10-20 times by sequentially using 400ml of anhydrous ethanol and 350ml of distilled water, obtaining high-quality magnetic graphene oxide precipitate, and then carrying out vacuum drying on the high-quality magnetic graphene oxide precipitate to obtain magnetic graphene oxide;

s2: preparing a polyethyleneimine matrix:

a, preparing polyethyleneimine emulsion: mixing 8-10 mL of 20% polyethyleneimine water solution, 10-12 mL of liquid paraffin and 2mL of polyvinyl alcohol pore-forming agent in percentage by mass, adding a surfactant into the mixed solution to form a mixed solution, wherein the final concentration of the surfactant is 0.04-0.06 g/mL, putting the mixed solution into ultrasonic equipment for ultrasonic mixing, and controlling the ultrasonic mixing time to be 2-3min to form polyethyleneimine emulsion;

b pre-crosslinking polymerization reaction: taking 15-20 mL of the polyethyleneimine emulsion, slowly dropwise adding 15-20 mL of a cross-linking agent under the action of magnetic stirring, and pre-crosslinking for 2-3 hours;

c, crosslinking polymerization reaction: under the condition of magnetic stirring, slowly dropwise adding 5-10 mL of sodium hydroxide solution into 20mL of pre-crosslinked mixture prepared by the pre-crosslinking polymerization reaction, controlling the concentration of the sodium hydroxide solution to be 1.0-2.0 mol/L, after the reaction is finished, performing centrifugal treatment on the mixture, and centrifuging to remove supernatant to obtain a polyethyleneimine matrix;

s3: preparing polyethyleneimine modified magnetic graphene oxide: weighing 20-40mg of magnetic graphene oxide, dissolving the magnetic graphene oxide in 20-40ml of phosphate buffer solution with the pH value of 7.4, then putting the mixed solution into ultrasonic equipment for ultrasonic mixing, and controlling the ultrasonic mixing time to be 5-10min to ensure that the mixed solution is uniformly dispersed; then sequentially adding 200mg of carbodiimide hydrochloride, 50-117mg of N-hydroxysuccinimide and 400mg of polyethyleneimine matrix into the mixed solution, uniformly stirring, filtering to obtain a filter cake, repeatedly washing the filter cake for 10 times by using 1000ml of ultrapure water to obtain a polyethyleneimine modified magnetic graphene oxide precipitate, and then carrying out vacuum drying on the polyethyleneimine modified magnetic graphene oxide precipitate to finally obtain polyethyleneimine modified magnetic graphene oxide;

s4: preparing magnetic graphene oxide modified by polyethyleneimine loaded with folic acid: weighing 5-20mg of polyethyleneimine modified magnetic graphene oxide, dissolving in 5-20ml of ultrapure water, adding 1-4mg of silane coupling agent and 5-20mg of folic acid powder, placing the mixed solution into ultrasonic equipment for ultrasonic mixing, and controlling the ultrasonic mixing time to be 1-2 hours to obtain the polyethyleneimine modified magnetic graphene oxide loaded with folic acid.

2. The method according to claim 1, wherein in S2, the addition amount of the cross-linking agent is the same as the addition amount of the polyethyleneimine emulsion.

3. The method according to claim 1, wherein in S2, the polyethyleneimine matrix is obtained by washing with isopropanol, dehydrated ether, and distilled water for 5-10 times.

4. The method for preparing magnetic graphene oxide capable of adsorbing beta-amyloid according to claim 1, wherein the temperature of vacuum drying of S3 is controlled to be 40-60 ℃, and the drying time is 1-2 days.

5. The method for preparing magnetic graphene oxide capable of adsorbing amyloid beta according to claim 1, wherein in S4, the silane coupling agent is 3-aminopropyltriethoxysilane.

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