Adsorptive Properties of Graphene Oxide and Its Promoting Effect of Nerve Regeneration

Author:Niu Qing Fei

Supervisor:zuo xiao jie


Degree Year:2017





Objective:Nanobiomedicine is a science with many discipline overlapping,the rise of nanobiomedicine to the development of bioscience and promoting human health plays an important role.Graphene is a famous nanomaterial discovered in 2004.It has been widely used because of its excellent adsorptive properties.Graphene oxide,as a derivative of graphene,has the characteristics of the super adsorptive properties.It also has a large number of functional groups such as hydroxyl,epoxy and carboxyl.Graphene oxide overcomes the shortcomings of the graphene such as clustering and difficult to dissolve in water.Graphene oxide shows better biological compatibility and stability in aqueous solution.It can be chemically functionalized easily to improve these features.In our study,graphene oxide was first used to adsorb bovine serum albumin(BSA)and vitamin B12(VB12).Then the functionalized nanocomposite was characterized.We improved the properties of graphene oxide and further studied the drug-carrier property of graphene oxide.Also we discovered the function of graphene oxide in promoting nerve regeneration by adsorbing vitamin B12 with it and applied it together with the acellular nerve allograft(ANA)in the injured sciatic nerve of rat.Protein is the most important biological macromolecule in human.It’s a product expressed by gene and execute important functions in vital activities as the smallest bioactive unit in life.Protein participated in all biological reactions and activities in human body.Serum albumin is the most abundant protein in serum,which accounts for 50%of total serum protein.It serves an important storage and transportation role as a carrier protein because of its high affinity to other molecules.Ji Xiufeng discovered albumin can be an excellent carrier for drugs.He studied the interaction among drug,nanomaterial and BSA using fluorescence spectrometry,ultraviolet absorption spectrometry and resonance Scattering Spectrometry.There are two ways to deliver the drug with the carrier protein.First approach is to create a molecular connection between the drug and albumin carrier aka the chemical-coupled albumin drug.Second approach is to encapsulate the drug with albumin nanoparticles through physical interactions between protein and drug.As nanotechnology develops rapidly,more and more research about it has been carried out and applied in biotechnology.The study of albumin nanoparticle drug delivery system has important clinical significance and development prospect with its new approaches to clinical drug development.Vitamin B12(VB12),also called cobalamin,is a class of chemically related compounds that contains cobalt in the corrin ring.It is the largest and most complicated vitamin so far.Also it’s the only vitamin contains metal ion.It’s also called the red vitamin because crystalized VB12 appears red.It has been proven that VB12 has therapeutic effects in treatment of many neurological diseases.Now it is a common medicine in clinical treatment of neurological diseases.Our team has been studying the morphological basis and regeneration mechanism theory of peripheral nerve injury,looking for substitute of large segment peripheral nerve injury.And we have received acellular nerve allograft and xenograft materials.Experiment proved that the nerve scaffold has can promote the nerve regeneration,the muscle reconstruction and the movement recovery with good biocompatibility.On this basis,this research will investigate the promoting effect of peripheral nerve regeneration of graphene oxide which is a new kind of nanobiological materials.Graphene oxide is a nanomaterial with high histocompatibility.It can adsorb VB12and modify the ANA by coating the ANA with a layer of drug-coupled functional nanoparticles.GO has excellent electrical conductivity.The conductivity of nerve depends on the electrical signal.We bridged the 10mm defect of injured sciatic nerve of rat with GO/ANA.After 11 weeks we assessed the action potentials of sciatic nerve,the growth associated protein-43(GAP43)expression,the growth of axon,the muscle rehabilitation of the operative side of rats in both GO/ANA group and ANA group.And the GO/ANA group shows significance in promoting nerve regeneration comparing to that of the ANA group.Methods:1.Preparation of graphene oxide with improved Hammer’s method and GO was characterized under electronic microscope.2.Functional groups in the GO samples were detected by Fourier transform infrared spectrometry.3.The optimum conditions for the adsorption of VB12 and BSA by nanomaterial was analyzed through single factor experiment method.With energy dispersive spectrum analysis,the element composition of the material can be analyzed to confirm the success of adsorption.4.GO-VB12/ANA was prepared by shake mixing and used to bridge the injured sciatic nerve by surgery.The effect of GO-VB12/ANA in promoting the regeneration of nerve was assessed by comparing with the control group.5.GAP43 expression was determined by immunofluorescence.Results:1.Under the scanning electron microscope,graphene oxide showed the single layer structure with folds and curls.Its water solution was brown after ultrasonic treatment.The absorption peaks of the carbonyl,hydroxyl and carboxyl groups were detected by Fourier transform infrared spectrometer which proved that there were many oxygen groups on the surface of GO.2.The optimal adsorption condition of the graphene oxide to BSA is 2.0g/L,shaking for 60 min at pH 5.The theoretical saturating adsorption of GO to BSA is 7.536g/g.3.The optimal adsorption condition of graphene oxide to VB12 is 6mg of GO in50mg/L of VB12,shaking for 70 min at pH 6.The theoretical saturating adsorption is21.51mg/g.4.GO-VB12/ANA repaired injured sciatic nerve.The nerve action potential,ratio of wet weight of gastrocnemius muscle,expression of GAP-43 in nerve fiber are significantly higher in GO-VB12/ANA group than that of the ANA group,close to the normal value without injuries.Data was shown as mean±standard deviation()and processed with SPSS11.0.Analysis of Variance was used to compare between groups.The two mean values were compared with t student test.α=0.05,when P<0.05,there is a significant difference.Conclusions:1.Graphene oxide can adsorb BSA and VB12.The optimal adsorption condition varies with different adsorbent for the same material.With the same adsorbent,the optimal adsorption condition also varies with different materials.2.ANA coated with graphene oxide coupled with VB12 can repair the injured sciatic nerve and promote the regeneration of nerve.Our study provides an experimental basis for clinical application of graphene oxide nanomaterial.