Nanometer material protective agent for cell preservation and preparation method thereof

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses a nano-material protective agent for cell preservation and a preparation method. The nanomaterial protective agent is prepared from the following raw materials in parts by weight: 0.5 to 2 parts of functionalized graphene oxide nanosheets, 0.3 to 1.5 parts of an amphiphilic block copolymer, 4 to 6 parts of trehalose, 1 to 3 parts of poly (vinyl pyrrolidone), 0.2 to 0.5 part of glutathione, 2 to 4 parts of polyethylene glycol and 83 to 92 parts of a phosphate buffer solution. The functionalized graphene oxide nanosheet is a graphene oxide nanosheet of which the surface is grafted with polyvinyl alcohol. The nano-material protective agent can reduce cell damage caused by reagents in the cell preservation process and improve the cell survival rate.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and particularly relates to a nanomaterial protectant for cell preservation and a preparation method thereof. Background Art

[0002] Cell preservation technology is an important foundation in the fields of biomedicine, regenerative medicine, and clinical treatment. Its goal is to maintain cell viability, function, and genetic stability under low-temperature or normal-temperature conditions. Traditional cell preservation methods mainly rely on cryoprotectants combined with programmed freezing technology, or achieve rapid solidification through vitrification preservation.

[0003] In related technologies, CN110839612A discloses a cord blood hematopoietic stem cell preservation solution. By weight, the raw materials include: 10-18 parts of low molecular weight dextran, 6-18 parts of dimethyl sulfoxide, 65-80 parts of DMEM medium, 4-8 parts of glycerol, 0.05-0.2 parts of folic acid, 0.1-0.5 parts of mannan oligosaccharide, and 1-5 parts of phosphatidylserine; the weight ratio range of the dimethyl sulfoxide, mannan oligosaccharide, and phosphatidylserine is 20-30:1:8-15.

[0004] However, due to the use of reagents such as dimethyl sulfoxide, during cell preservation, trace amounts of DMSO may penetrate the cell membrane, causing osmotic imbalance or directly damaging the cell membrane structure, resulting in a decrease in cell viability. Summary of the Invention

[0005] In order to improve cell viability, the present application provides a nanomaterial protectant for cell preservation and a preparation method thereof.

[0006] In the first aspect, a nanomaterial protectant for cell preservation provided by the present application adopts the following technical solution:

[0007] A nanomaterial protectant for cell preservation includes the following raw materials by weight: 0.5-2 parts of functionalized graphene oxide nanosheets, 0.3-1.5 parts of amphiphilic block copolymer, 4-6 parts of trehalose, 1-3 parts of poly(vinylpyrrolidone), 0.2-0.5 parts of glutathione, 2-4 parts of polyethylene glycol, and 83-92 parts of phosphate buffer solution; the functionalized graphene oxide nanosheets are graphene oxide nanosheets grafted with polyvinyl alcohol on the surface.

[0008] By adopting the above technical solutions, in the functionalized graphene oxide nanosheets, the hydroxyl groups of polyvinyl alcohol bind water molecules through hydrogen bonds to inhibit ice crystal nucleation; the two-dimensional sheets of graphene oxide divide the ice domain to limit ice crystal growth. After the functionalized graphene oxide nanosheets wrap cells, the π-π conjugation of graphene oxide adsorbs cell membrane phospholipids, and the flexible chains of polyvinyl alcohol form a "molecular brush" to reduce mechanical damage. The amphiphilic block copolymer can reduce the aggregation of functionalized graphene oxide nanosheets, ensure the stability of the micelles, and promote the interaction between cells and functionalized graphene oxide nanosheets. Trehalose forms a glassy matrix to reduce the free water content. The pyrrolidone groups of poly(vinylpyrrolidone) can form a hydrogen bond network with trehalose to enhance the ice crystal inhibition effect. Poly(vinylpyrrolidone) can also adsorb on the surface of ice crystals and cooperate with functionalized graphene oxide nanosheets to promote the reduction of ice crystal size and prevent ice crystals from piercing the cell membrane. Polyethylene glycol adjusts the osmotic pressure to be close to the physiological value, glutathione neutralizes ·OH and H2O2, and phosphate buffer maintains the pH and ionic balance. Therefore, the nanomaterial protectant with the above raw material ratio can reduce the damage of reagents to cells during cell preservation and improve the cell survival rate.

[0009] In a specific feasible embodiment, the amphiphilic block copolymer is any one of PEO-PPO-PEO triblock copolymer or PCL-PEG block copolymer.

[0010] By adopting the above technical solutions, when choosing the PEO-PPO-PEO triblock copolymer, the PEO chain segment reduces the direct contact between the nanomaterial and the cell membrane through steric hindrance, avoiding mechanical damage. Moreover, the PEO chain adsorbs on the surface of the nanomaterial to prevent its aggregation. The ether bond of the PEO chain forms a hydrogen bond with the hydroxyl group of trehalose to synergistically inhibit ice crystal nucleation and growth. When choosing the PCL-PEG block copolymer, the flexibility of PEG is combined with the rigidity of PCL to form a protective layer with moderate mechanical strength, reducing membrane rupture during the freeze-thaw process. The PCL segment can also insert into the cell membrane phospholipid layer to inhibit the phase transition of membrane lipids at low temperature.

[0011] In a specific feasible embodiment, the nanomaterial protectant for cell preservation further includes sphingomyelin.

[0012] By adopting the above technical solutions, sphingomyelin metabolites can activate the cell membrane repair pathway and accelerate the repair of membrane damage after resuscitation. Moreover, the rigid structure of sphingomyelin hinders the binding of ice crystals to the membrane surface and reduces the risk of ice crystals piercing the membrane.

[0013] In a specific feasible embodiment, the nanomaterial protectant for cell preservation further includes adenosine triphosphate and sodium pyruvate.

[0014] By adopting the above technical solution, adenosine triphosphate can rapidly replenish the intracellular ATP pool during the resuscitation stage, alleviating the energy depletion during cryopreservation. Pyruvate bypasses the rate-limiting step of glycolysis and directly enters the mitochondria to generate acetyl coenzyme A, accelerating the TCA cycle. ATP and sodium pyruvate cooperate to balance energy supply and metabolic demands, increasing cell survival rate.

[0015] In a specific feasible embodiment, the nanomaterial protectant for cell preservation further includes mannitol.

[0016] By adopting the above technical solution, mannitol can not only prevent the hydrolysis of ATP but also balance the osmotic pressure, preventing the excessive osmotic pressure that may be caused by sodium pyruvate.

[0017] In a second aspect, a preparation method of a nanomaterial protectant for cell preservation provided by this application adopts the following technical solution:

[0018] A preparation method of a nanomaterial protectant for cell preservation includes the following steps:

[0019] Add functionalized graphene oxide nanosheets into water and disperse evenly to obtain an F-GO dispersion;

[0020] Add amphiphilic block copolymer into the F-GO dispersion, heat to 40 - 45 °C, and stir evenly to obtain a mixed solution;

[0021] Dissolve trehalose and poly(vinylpyrrolidone) in phosphate buffer solution, heat to 37 - 42 °C and stir evenly, then add the mixed solution and disperse evenly to obtain a homogeneous suspension;

[0022] Add glutathione and polyethylene glycol into the homogeneous suspension and stir evenly under dark conditions to obtain the nanomaterial protectant for cell preservation.

[0023] By adopting the above technical solution, heating to 40 - 45 °C, which is close to the critical micelle temperature of the amphiphilic block copolymer, can promote the hydrophilic chain segments of the amphiphilic block copolymer to wrap the surface of the functionalized graphene oxide nanosheets, enhancing its dispersion stability. Phosphate buffer solution is used to maintain physiological pH and ionic strength, avoiding cell osmotic pressure imbalance. Heating at 37 - 42 °C promotes the dissolution of trehalose and poly(vinylpyrrolidone), avoiding crystallization precipitation caused by low temperature. Dark conditions prevent the photolysis of glutathione, ensuring its reducing ability. Glutathione binds to the carboxyl groups on the surface of the functionalized graphene oxide nanosheets through hydrogen bonds to form a dynamic antioxidant network, and PEG-400 adjusts the solution viscosity.

[0024] In a specific feasible embodiment, the synthesis steps of the functionalized graphene oxide nanosheets are as follows:

[0025] Disperse graphene oxide in deionized water until evenly dispersed to obtain a GO dispersion with a concentration of 1 - 1.5 mg / mL, centrifuge, and collect the supernatant;

[0026] Heat DMSO to 60 - 65 °C, add polyvinyl alcohol to obtain a polyvinyl alcohol solution with a concentration of 10 - 12 mg / mL;

[0027] Mix the supernatant with the polyvinyl alcohol solution, add EDC and NHS with a molar ratio of 1:(1 - 1.4), react under nitrogen protection at 50 - 55 °C for 20 - 24 h, then transfer to a dialysis bag and dialyze with deionized water for 72 - 84 h, changing the water every 6 - 7 h to obtain functionalized graphene oxide nanosheets.

[0028] By adopting the above technical solutions, a GO concentration of 1 - 1.5 mg / mL balances the dispersion stability and reaction efficiency, avoiding re - aggregation caused by too high a concentration. A polyvinyl alcohol concentration of 10 - 12 mg / mL can provide a sufficient reactant density to promote the grafting efficiency with GO. At the same time, it avoids too high a solution viscosity from hindering molecular diffusion. Heating at 60 - 65 °C can accelerate the movement of polyvinyl alcohol chain segments, improve the reaction activity of hydroxyl groups, and reduce the dissolution time. The EDC / NHS molar ratio of 1:1 - 1.4 can maximize the carboxyl activation efficiency and reduce the generation of by - products. Under nitrogen protection at 50 - 55 °C, it can accelerate the reaction kinetics while avoiding excessive reduction of GO or degradation of polyvinyl alcohol. Dialysis removes unreacted EDC, NHS, by - products, and residual DMSO. 72 - 84 h of dialysis combined with frequent water changes achieves gradient impurity removal and avoids concentration rebound.

[0029] In summary, the present application has the following beneficial effects:

[0030] 1. By using the nanomaterial protective agent with the above - mentioned raw material ratio, the present application can reduce the damage of reagents to cells during cell preservation and improve cell survival rate.

[0031] 2. Sphingomyelin is preferably used in the present application, which can accelerate the repair of membrane damage after resuscitation and reduce the risk of ice crystals piercing the membrane.

[0032] 3. The method of the present application can promote the hydrophilic chain segments of the amphiphilic block copolymer to wrap the surface of the functionalized graphene oxide nanosheets, enhancing their dispersion stability. Detailed implementation mode

[0033] PEO-PPO-PEO triblock copolymer, CAS No.: 9003-11-6, purchased from Changzhou Angxing New Carbon Materials Co., Ltd. Glutathione, CAS No.: 70-18-8, purchased from Guangdong Mingcheng Biotechnology Co., Ltd. PCL-PEG block copolymer was purchased from Hangzhou New Qiao Biotechnology Co., Ltd. Phosphate buffer (PBS), model number FORLAB FL10210. Sphingomyelin, CAS No.: 6254-89-3, purchased from Chengdu Purifa Technology Development Co., Ltd.

[0034] The present application will be further described in detail below in conjunction with examples and comparative examples.

[0035] Examples

[0036] Example 1

[0037] This example provides a nanomaterial protectant for cell preservation, including the following raw materials: 0.5 g of functionalized graphene oxide nanosheets, 0.3 g of PEO-PPO-PEO triblock copolymer, 4 g of trehalose, 1 g of poly(vinylpyrrolidone), 0.2 g of glutathione, 2 g of polyethylene glycol, and 92 g of phosphate buffer.

[0038] The functionalized graphene oxide nanosheets were synthesized according to the following steps:

[0039] Graphene oxide was added to deionized water and ultrasonically oscillated until evenly dispersed to obtain a 1 mg / mL GO dispersion. Then, the GO dispersion was centrifuged at 5000 rpm to remove unpeeled aggregates, and the supernatant was collected.

[0040] After heating DMSO to 60 °C, polyvinyl alcohol was added to obtain a 10 mg / mL polyvinyl alcohol solution;

[0041] The supernatant and the polyvinyl alcohol solution were mixed at a volume ratio of 10:1, and EDC and NHS with a molar ratio of 1:1 were added. Then, after reacting under nitrogen protection at 50 °C for 24 h, it was transferred to a dialysis bag and dialyzed with deionized water for 72 h, changing the water every 6 h, to obtain functionalized graphene oxide nanosheets.

[0042] This example also provides a preparation method for a nanomaterial protectant for cell preservation, including the following steps:

[0043] The functionalized graphene oxide nanosheets were added to water and evenly dispersed to obtain an F-GO dispersion.

[0044] The PEO-PPO-PEO triblock copolymer was added to the F-GO dispersion, heated to 40 °C, and stirred evenly to obtain a mixed solution.

[0045] Add trehalose and poly(vinylpyrrolidone) to phosphate buffer solution, then heat to 37 °C, stir to dissolve, and after stirring evenly, add the mixed solution dropwise. After the addition is completed, ultrasonically vibrate until evenly dispersed to obtain a homogeneous suspension.

[0046] Add glutathione and polyethylene glycol to the homogeneous suspension and stir evenly under light-shielded conditions to obtain a nanomaterial protective agent for cell preservation.

[0047] Example 2

[0048] The difference between this example and Example 1 is only that the nanomaterial protective agent for cell preservation includes the following raw materials: 2 g of functionalized graphene oxide nanosheets, 1.5 g of PEO-PPO-PEO triblock copolymer, 6 g of trehalose, 3 g of poly(vinylpyrrolidone), 0.5 g of glutathione, 4 g of polyethylene glycol, and 83 g of phosphate buffer solution.

[0049] Example 3

[0050] The difference between this example and Example 1 is only that the nanomaterial protective agent for cell preservation includes the following raw materials: 1.2 g of functionalized graphene oxide nanosheets, 1 g of PEO-PPO-PEO triblock copolymer, 5 g of trehalose, 2 g of poly(vinylpyrrolidone), 0.3 g of glutathione, 3 g of polyethylene glycol, and 87.5 g of phosphate buffer solution.

[0051] Example 4

[0052] The difference between this example and Example 1 is only that the PEO-PPO-PEO triblock copolymer is replaced with an equal amount of PCL-PEG block copolymer.

[0053] Example 5

[0054] The difference between this example and Example 1 is only that the functionalized graphene oxide nanosheets are synthesized according to the following steps:

[0055] Add graphene oxide to deionized water and ultrasonically vibrate until evenly dispersed to obtain a 1.5 mg / mL GO dispersion. Then centrifuge the GO dispersion at 5000 rpm to remove unpeeled aggregates and collect the supernatant.

[0056] After heating DMSO to 65 °C, add polyvinyl alcohol to obtain a polyvinyl alcohol solution with a concentration of 12 mg / mL;

[0057] Mix the supernatant and the polyvinyl alcohol solution at a volume ratio of 10:1, add EDC and NHS with a molar ratio of 1:1.4, and then, after holding and reacting at 55 °C under nitrogen protection for 20 h, transfer it to a dialysis bag and dialyze with deionized water for 84 h, changing the water every 7 h to obtain functionalized graphene oxide nanosheets.

[0058] Example 6

[0059] The difference between this example and Example 1 is only that the nanomaterial protectant for cell preservation includes the following raw materials: 0.5 g of functionalized graphene oxide nanosheets, 0.3 g of PEO-PPO-PEO triblock copolymer, 4 g of trehalose, 1 g of poly(vinylpyrrolidone), 0.2 g of glutathione, 2 g of polyethylene glycol, 91.7 g of phosphate buffer solution, and 0.3 g of sphingomyelin. In the preparation method of the nanomaterial protectant for cell preservation, glutathione, polyethylene glycol, and sphingomyelin are added to the homogeneous suspension and stirred evenly under dark conditions to obtain the nanomaterial protectant for cell preservation.

[0060] Example 7

[0061] The difference between this example and Example 1 is only that the nanomaterial protectant for cell preservation includes the following raw materials: 0.5 g of functionalized graphene oxide nanosheets, 0.3 g of PEO-PPO-PEO triblock copolymer, 4 g of trehalose, 1 g of poly(vinylpyrrolidone), 0.2 g of glutathione, 2 g of polyethylene glycol, 91.4 g of phosphate buffer solution, 0.3 g of adenosine triphosphate, and 0.3 g of sodium pyruvate. In the preparation method of the nanomaterial protectant for cell preservation, glutathione, polyethylene glycol, adenosine triphosphate, and sodium pyruvate are added to the homogeneous suspension and stirred evenly under dark conditions to obtain the nanomaterial protectant for cell preservation.

[0062] Example 8

[0063] The difference between this example and Example 1 is only that the nanomaterial protectant for cell preservation includes the following raw materials: 0.5 g of functionalized graphene oxide nanosheets, 0.3 g of PEO-PPO-PEO triblock copolymer, 4 g of trehalose, 1 g of poly(vinylpyrrolidone), 0.2 g of glutathione, 2 g of polyethylene glycol, 91.7 g of phosphate buffer solution, and 0.3 g of mannitol. In the preparation method of the nanomaterial protectant for cell preservation, glutathione, polyethylene glycol, and mannitol are added to the homogeneous suspension and stirred evenly under dark conditions to obtain the nanomaterial protectant for cell preservation.

[0064] Example 9

[0065] The difference between this example and Example 1 is only that the nanomaterial protectant for cell preservation includes the following raw materials: 0.5 g of functionalized graphene oxide nanosheets, 0.3 g of PEO-PPO-PEO triblock copolymer, 4 g of trehalose, 1 g of poly(vinylpyrrolidone), 0.2 g of glutathione, 2 g of polyethylene glycol, 91.1 g of phosphate buffer solution, 0.3 g of adenosine triphosphate, 0.3 g of sodium pyruvate, and 0.3 g of mannitol. In the preparation method of the nanomaterial protectant for cell preservation, glutathione, polyethylene glycol, adenosine triphosphate, sodium pyruvate, and mannitol are added to the homogeneous suspension and stirred evenly under dark conditions to obtain the nanomaterial protectant for cell preservation.

[0066] Example 10

[0067] The difference between this example and Example 1 is only that the nanomaterial protectant for cell preservation includes the following raw materials: 0.5 g of functionalized graphene oxide nanosheets, 0.3 g of PEO-PPO-PEO triblock copolymer, 4 g of trehalose, 1 g of poly(vinylpyrrolidone), 0.2 g of glutathione, 2 g of polyethylene glycol, 90.8 g of phosphate buffer solution, 0.3 g of sphingomyelin, 0.3 g of adenosine triphosphate, 0.3 g of sodium pyruvate, and 0.3 g of mannitol. In the preparation method of the nanomaterial protectant for cell preservation, glutathione, polyethylene glycol, sphingomyelin, adenosine triphosphate, sodium pyruvate, and mannitol are added to the homogeneous suspension and stirred evenly under dark conditions to obtain the nanomaterial protectant for cell preservation.

[0068] Example 11

[0069] The difference between this example and Example 1 is only that the preparation method of the nanomaterial protectant for cell preservation includes the following steps:

[0070] Add the functionalized graphene oxide nanosheets into water and disperse evenly to obtain the F-GO dispersion.

[0071] Add the PEO-PPO-PEO triblock copolymer into the F-GO dispersion, heat to 45 °C, and stir evenly to obtain a mixed solution.

[0072] Add trehalose and poly(vinylpyrrolidone) into the phosphate buffer solution, then heat to 42 °C, stir and dissolve, after stirring evenly, dropwise add the mixed solution, and after the dropping is completed, perform ultrasonic oscillation until evenly dispersed to obtain a homogeneous suspension.

[0073] Add glutathione and polyethylene glycol into the homogeneous suspension and stir evenly under dark conditions to obtain the nanomaterial protectant for cell preservation.

[0074] Comparative example

[0075] Comparative example 1

[0076] The only difference between this comparative example and Example 1 is that an equal amount of graphene oxide is used to replace the functionalized graphene oxide nanosheets.

[0077] Comparative Example 2

[0078] The only difference between this comparative example and Example 1 is that an equal amount of phosphate buffer solution is used to replace the functionalized graphene oxide nanosheets.

[0079] Comparative Example 3

[0080] The only difference between this comparative example and Example 1 is that an equal amount of phosphate buffer solution is used to replace the amphiphilic block copolymer.

[0081] Comparative Example 4

[0082] The only difference between this comparative example and Example 1 is that an equal amount of phosphate buffer solution is used to replace trehalose.

[0083] Comparative Example 5

[0084] The only difference between this comparative example and Example 1 is that an equal amount of phosphate buffer solution is used to replace poly(vinylpyrrolidone).

[0085] Comparative Example 6

[0086] The only difference between this comparative example and Example 1 is that an equal amount of phosphate buffer solution is used to replace glutathione.

[0087] Comparative Example 7

[0088] The only difference between this comparative example and Example 1 is that an equal amount of phosphate buffer solution is used to replace polyethylene glycol.

[0089] Performance detection test

[0090] For the nanomaterial protectants for cell preservation in Examples 1 - 11 and Comparative Examples 1 - 7, the detection is carried out according to the following steps:

[0091] Experimental group:

[0092] Digest and centrifuge the mesenchymal stem cells (1000 rpm, 5 min), discard the supernatant, wash twice with PBS, and resuspend the treated mesenchymal stem cells in the nanomaterial protectant for cell preservation according to the cell density of 1×10 6 cells / mL, and then transfer them to cryotubes for storage. Place the cryotubes in a programmable freezer, cool them to -80°C at a rate of -1°C / min, and transfer them to liquid nitrogen for storage.

[0093] For each nanomaterial cryoprotectant used for cell preservation, two groups of experiments were set up according to the above steps, one group was preserved for 7 days and the other group was preserved for 14 days.

[0094] Control group 1:

[0095] Digest and centrifuge the mesenchymal stem cells (1000 rpm, 5 min), discard the supernatant, wash twice with PBS, and resuspend the treated mesenchymal stem cells in the medium containing 10% DMSO at a cell density of 1×10 6 cells / mL, and then transfer them to cryotubes for preservation. Then place the cryotubes in a programmable freezer and cool them to -80°C at a rate of -1°C / min, and transfer them to liquid nitrogen for storage.

[0096] Set up two groups of experiments according to the above steps, one group was preserved for 7 days and the other group was preserved for 14 days.

[0097] Control group 2:

[0098] Digest and centrifuge the mesenchymal stem cells (1000 rpm, 5 min), discard the supernatant, wash twice with PBS, and resuspend the treated mesenchymal stem cells in PBS at a cell density of 1×10 6 cells / mL, and then transfer them to cryotubes for storage. Then place the cryotubes in a programmable freezer and cool them to -80°C at a rate of -1°C / min, and transfer them to liquid nitrogen for storage.

[0099] Set up two groups of experiments according to the above steps, one group was preserved for 7 days and the other group was preserved for 14 days.

[0100] Detect the survival rate:

[0101] After the preservation is completed, place the cryotubes in a 37°C water bath and quickly thaw for 1 min to obtain the resuscitated cell suspension.

[0102] Take 10 μL of the resuscitated cell suspension, mix it with an equal volume of 0.4% trypan blue staining solution, let it stand for 3 min, and count according to a hemocytometer to calculate the survival rate: Survival rate = number of live cells (unstained) / total number of cells × 100%.

[0103] The detection results are shown in Table 1.

[0104] Table 1

[0105]

[0106]

[0107] Combined with Example 1 and Comparative Examples 1-2 and Table 1, it can be seen that compared with Example 1, the cell survival rates after 7 days of preservation and after 14 days of preservation in Comparative Example 1 are both significantly reduced. For Comparative Example 2, the cell survival rates after 7 days of preservation and after 14 days of preservation are significantly smaller, and are less than those in Comparative Example 1. This shows that using the nanomaterial protectant for cell preservation in Example 1 helps to improve the cell survival rate.

[0108] Combined with Examples 1-11 and Comparative Examples 1-7 and Table 1, it can be seen that the cell survival rates after 7 days of preservation in Comparative Examples 1-7 are all below 90%, and the cell survival rates after 14 days of preservation are all below 86%. The cell survival rates after 7 days of preservation in Examples 1-11 are all above 96%, and the cell survival rates after 14 days of preservation are all above 93%. This shows that using the nanomaterial protectants for cell preservation in Examples 1-11 can all improve the cell survival rate.

[0109] This specific embodiment is only an explanation of the present application and does not limit the present application. Those skilled in the art can make modifications to this embodiment without creative contributions according to needs after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A nanomaterial protective agent for cell preservation, characterized in that, It comprises raw materials in the following parts by weight: 0.5 - 2 parts of functionalized graphene oxide nanosheets, 0.3 - 1.5 parts of amphiphilic block copolymer, 4 - 6 parts of trehalose, 1 - 3 parts of poly(vinylpyrrolidone), 0.2 - 0.5 parts of glutathione, 2 - 4 parts of polyethylene glycol, and 83 - 92 parts of phosphate buffer solution; the functionalized graphene oxide nanosheets are graphene oxide nanosheets grafted with polyvinyl alcohol on the surface.

2. The nanomaterial protective agent for cell preservation according to claim 1, characterized in that, The amphiphilic block copolymer is any one of PEO-PPO-PEO triblock copolymer or PCL-PEG block copolymer.

3. The nano-material protective agent for cell preservation according to claim 1, characterized in that, The nanomaterial protectant for cell preservation further comprises sphingomyelin.

4. The nanomaterial protective agent for cell preservation according to claim 1, characterized in that, The nanomaterial protectant for cell preservation further comprises adenosine triphosphate and sodium pyruvate.

5. The nanomaterial protective agent for cell preservation according to claim 4, wherein, The nanomaterial protectant for cell preservation further comprises mannitol.

6. A preparation method of the nanomaterial protective agent for cell preservation according to any one of claims 1-5, characterized in that, It comprises the following steps: Add the functionalized graphene oxide nanosheets into water, disperse evenly to obtain an F-GO dispersion. Add the amphiphilic block copolymer into the F-GO dispersion, heat to 40 - 45 °C, and stir evenly to obtain a mixture. Dissolve trehalose and poly(vinylpyrrolidone) in the phosphate buffer solution, heat to 37 - 42 °C and stir evenly, then add the mixture and disperse evenly to obtain a homogeneous suspension. Add glutathione and polyethylene glycol into the homogeneous suspension, stir evenly under light protection to obtain the nanomaterial protectant for cell preservation.

7. The preparation method of the nanomaterial protective agent for cell preservation according to claim 6, wherein, The synthesis steps of the functionalized graphene oxide nanosheets are as follows: Disperse graphene oxide in deionized water, disperse evenly to obtain a GO dispersion with a concentration of 1 - 1.5 mg / mL, centrifuge, and collect the supernatant. Heat DMSO to 60 - 65 °C, add polyvinyl alcohol to obtain a polyvinyl alcohol solution with a concentration of 10 - 12 mg / mL. Mix the supernatant with the polyvinyl alcohol solution, add EDC and NHS with a molar ratio of 1:(1 - 1.4), react at 50 - 55 °C under nitrogen protection for 20 - 24 h, then transfer to a dialysis bag and dialyze with deionized water for 72 - 84 h, changing the water every 6 - 7 h to obtain the functionalized graphene oxide nanosheets.

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