Research On Surface Organic Composite Graphene Oxide And Application In Cell Cryopreservation

Cryopreservation techniques allow for the long-term storage of organs,tissues,cells and other biological materials by significantly reducing or even stopping chemical and biological reactions in living cells.However,the formation,growth and recrystallization of ice crystals during cryopreservation will cause fatal mechanical damage to cells.Therefore,how to effectively regulate and inhibit ice crystals,reduce freezing damage,and improve cryopreservation efficiency is a critical scientific problem that needs to be addressed in the current cryopreservation field.The two-dimensional nanomaterials themselves exhibit good ice recrystallization inhibition activity and can accelerate the rapid cell rewarming process by exhibiting efficient photothermal conversion ability during frozen cell rewarming.Therefore,it is of great theoretical significance and practical value to develop new composite materials based on 2D nanomaterials that can be used to inhibit ice crystallization,reduce freezing damage and improve cell survival during cell freezing and rapid rewarming in modern medical research.Based on the above research idea,this thesis combined the ice recrystallization inhibition activity and photothermal conversion ability of graphene oxide(GO)and polyvinyl alcohol(PVA),based on which a polyvinyl alcohol grafted graphene oxide ice control material was prepared and successfully applied to the cryopreservation and rapid resuscitation of Hela cells and A549 lung cancer cells.The details of the study are as follows:(1)Monolayer graphene oxide nanosheets with a size distribution of>100 nm were selected for the polyvinyl alcohol modification of graphene oxide using carbodiimide catalyzed esterification reaction.Moreover,to investigate the effects of different polymerization degrees and different polyvinyl alcohol doping ratios on the material properties,three different polymerization degrees of polyvinyl alcohol and three different doping ratios were used for the synthesis experiments.The morphological and structural characterization of the synthesized nine composite nanomaterials confirmed the successful synthesis of polyvinyl alcohol grafted graphene oxide(PVA-g-GO).(2)Ice recrystallization inhibition activity(IRI)tests were performed on the nine materials by the"cold bench freezing method",and the experimental results showed that the IRI activity of poly(vinyl alcohol)was the best at the polymerization degree of 1700～1800,and the best IRI activity was obtained when the mass ratio of graphene oxide to poly(vinyl alcohol)was 10:1 when PVA 17-88 was used.The IRI activity was best when PVA 17-88 type was used with a mass ratio of 10:1 between graphene oxide and polyvinyl alcohol.(3)Using a laser generator with a wavelength of 808 nm and a power density of 2W/cm~2,the photothermal conversion ability of the screened group of solutions with different concentrations of PVA-g-GO with the best ice recrystallization inhibition activity was tested,and it was found that PVA-g-GO exhibited a strong photothermal conversion ability,even at a concentration of 20μg/m L,and could be raised from room temperature to 82.8℃within 60 s.Even at a concentration of 20μg/m L,the PVA-g-GO could be elevated from room temperature to 82.8℃within 60 s.The temperature elevation effect was remarkable,and the photothermal conversion efficiency could reach～39.7%.Compared with some heavy metals and semiconductor photothermal conversion materials have obvious advantages,and also laid a good foundation for the subsequent work.(4)The MTT cytotoxicity test was performed on selected PVA-g-GO,and Hela cells and A549 lung cancer cells were still able to maintain more than 75%relative viability after co-incubation for 24 h.The experimental results indicated that PVA-g-GO had little effect on the cells.At the same time,the two cells were cryopreserved in PVA-g-GO cryoprotective agent solution and resuscitated by laser heating,and the results showed that the survival rate of both cells was above 75%after resuscitation.The development of this technology facilitates the efficient preservation of biomaterials of higher medical value and opens up ideas for subsequent research.