功能化氧化石墨烯的细胞相容性

[Article]物理化学学报(Wuli Huaxue Xuebao )Acta Phys.⁃Chim.Sin .2012,28(6),1520-1524JuneReceived:November 25,2011;Revised:March 11,2012;Published on Web:March 13,2012.∗Corresponding authors.YANG Rong,Email:yangr@;Tel:+86-10-82545616.HENG Cheng-Lin,Email:hengcl@.The project was supported by the National Natural Science Foundation of China (20911130229,21073047)and Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences,China (KJCX2.YW.M15).国家自然科学基金(20911130229,21073047)和中国科学院知识创新工程重要方向项目(KJCX2.YW.M15)资助ⒸEditorial office of Acta Physico ⁃Chimica Sinicadoi:10.3866/PKU.WHXB 201203131功能化氧化石墨烯的细胞相容性张晓1,2杨蓉2,*王琛2衡成林1,*(1北京理工大学物理学院,教育部簇科学重点实验室,北京100081;2国家纳米科学中心,中国科学院纳米生物效应与安全性重点实验室,北京100190)摘要:采用改进的Hummers 方法制备了纳米尺度的氧化石墨烯.对氧化石墨烯的表面进行羧基化,并连接上聚乙二醇(PEG)使其在细胞培养液中可溶并能稳定保存.采用透射电镜(TEM)、傅里叶变换红外(FTIR)光谱和zeta 电位测量等对修饰后的氧化石墨烯的结构和功能进行了表征.体外细胞实验表明PEG 修饰的氧化石墨烯在水中具有良好的可溶性,对A549细胞没有明显的毒性,在生物医药领域具有潜在的应用价值.关键词:氧化石墨烯;纳米材料;生物相容性;表面功能化中图分类号:O645Cell Biocompatibility of Functionalized Graphene OxideZHANG Xiao 1,2YANG Rong 2,*WANG Chen 2HENG Cheng-Lin 1,*(1Key Laboratory of Cluster Science of Ministry of Education,School of Physics,Beijing Institute of Technology,Beijing 100081,P .R.China ;2Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety,Chinese Academy of Sciences,National Center for Nanoscience and Technology,Beijing 100190,P .R.China )Abstract:We report on synthesis of nanoscale graphene oxide (NGO)by modified Hummers ’method.Synthesized NGO particles were surface functionalized by attaching carboxylic acid and polyethylene glycol groups to render them soluble in cell culture medium.The structures and properties of functionalized NGO were characterized by transmission electron microscopy (TEM),Fourier transform infrared (FTIR)spectroscopy,and zeta potential analyzer.Cell viability studies show that PEG-modified NGO particles are highly soluble and incur almost no cytotoxicity to A549cells,which suggest a great potential for the use of NGO in various biomedical applications.Key Words:Graphene oxide;Nanomaterials;Biocompatibility;Surface functionalization1IntroductionGraphene,a single layer of carbon atoms with excellent ther-mal,mechanical,and electrical properties,has attracted consid-erable attention in recent years.1-3Graphene oxide (GO)4-10is one of the most important graphene derivatives which contains aromatic regions randomly interspersed with oxidized aliphatic rings.These oxidized rings containing epoxide (C ―O ―C)and hydroxyl (C ―OH)groups,and the GO sheets terminated with both carbonyl (C ＝O)and carboxylic acid (―COOH)groups 5-7can provide reactive sites for chemical modification to obtain new derivatives for biology application.8-10It is known that many pharmaceutical ingredients are poorly solu-ble in water.As a result,their clinical applications are seriously influenced.Therefore,finding a nanoscale drug carrier is criti-cal in biology application.Recently,researchers started to ex-plore the ability of GO in attachment and delivery of aromatic,water insoluble drugs.Yang et al.11investigated loading doxo-rubicin hydrochloride,an anticancer drug,on GO sheets,and1520ZHANG Xiao et al.:Cell Biocompatibility of Functionalized Graphene Oxide No.6found that the loading ratio(mass ratio of loaded drug to carri-ers)of GO could reach200%,much higher than those of other nanocarriers such as nanoparticles that usually have a loading ratio lower than100%.Liuʹs group12studied the in vitro behav-iors of GO in animal experiments.However,before GO material can be used in biology,two main problems should be resolved.First,GO is known to dis-perse well in water but it generally aggregates in salt or other biological solutions.8-10Second,it is not easy to get GO sheets with uniform and suitable size.Size control or size separation on various length scales is necessary to suitably interface with biological systems in vitro or in vivo.12-14Here,we have employed a modified Hummersʹmethod15-18 to fabricate water-soluble nanoscale graphene oxide(NGO), and then performed surface functionalized modification to syn-thesize the PEG functionalized carboxylic acid modified na-noscale graphene oxide(NGO-COOH-PEG)structure.Finally, we have examined the solubility of NGO-COOH-PEG and cy-totoxicity of NGO-COOH-PEG to human lung adenocarcino-ma cells(A549)pared to carbon nanotubes15and nanohorns16loading drugs mainly via surface and tip,our modi-fied NGO has a large specific surface area and loads aromatic anticancer drugs via its two faces and edges,which makes it a promising material as a drug carrier substance.2Materials and methods2.1MaterialsGraphite flake(325mesh),polyethylene glycol(PEG), 1-(3-dimethylamlnopropyl)-3-ethylcarbodiimide hydrochloride (EDC)and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-um bromide(MTT)were supplied by Sigma-Aldrich Chemi-cal;Sulfuric acid(98%H2SO4),KMnO4,H2O2,NaOH, ClCH2COONa,and dimethyl sulfoxide(DMSO)were supplied by Sinopharm Chemical Reagent.All the reagents are of analyt-ical reagent grade and used without further purification.The A549were supplied by Beijing Union Medical College.2.2Synthesis of surface functionalized nanoscalegraphene oxideA modified Hummersʹmethod15-18was used to obtain graph-ite oxide from graphite flake,and then the graphite oxide was dispersed in ultra-pure water to form graphite oxide suspen-sion.The suspension was sonicated for3h so that the graphite oxide became mostly single layered.After that,the suspension was centrifuged for20min to remove undesired impurities, and then the GOs left in suspension are in nanoscale level.In order to attach PEG with NGO,―COOH functional group must be introduced to the NGO surface.First,the NGO suspension was prepared with a concentration of2g·L-1.Then, 0.06mol NaOH and0.03mol ClCH2COONa were added to the suspension and sonicated for2h.The hydroxyl,epoxide,and the carbonyl groups of NGO were reacted chemically and con-verted into―COOH groups upon sonicating the mixture.17Af-ter removing the salt and NaOH from the reacted residual by centrifuging against distilled water,the final product carboxyl-ic acid modified graphene oxide(NGO-COOH)was precipitat-ed and dispersed in water.The color of the NGO suspension changed from brown to black during the reaction,probably due to partial reduction of the NGO under the strong alkaline condi-tion.18After that,the PEG with a concentration of2g·L-1was add-ed to the NGO-COOH solution.The solution was sonicated with EDC for4h.Then the solution was stored for24h so that PEG-amine can combine the NGO-COOH with EDC.The sus-pension was centrifuged for several times.Finally,we got the PEG functionalized carboxylic acid modified graphene oxide (NGO-COOH-PEG).2.3CharacterizationThe structure of the asprepared NGO-COOH-PEG were characterized by transmission electron microscope(FEI Tecnai G220),Fourier transform infrared spectrometer(Perkin Elmer).2.4MTT assay for cell viability testA549cells were cultured in RPMI1640medium(Thermo Scientific)with10%(φ)fetal bovine serum(FBS)and100IU·mL-1penicillin and100µg·mL-1streptomycin,respectively. The culture plates were incubated at37°C in a humidified in-cubator containing5%CO2.The cell viability was tested using MTT assay,which is based on the mitochondrial conversion of tetrazolium salt.19The A549cells were seeded in96-well mi-croplates(Costar,Corning,NY)at densities of8000and5000 cells·well-1respectively in100μL RPMI1640medium(devel-oped by Moore et.al.at Roswell Park Memorial Institute)con-taining10%fetal bovine serum(FBS).After attachment for24 h,the cells were incubated with NGO-COOH-PEG at various concentrations(3.4,13.6,54.4,85.0,and102.0mg·L-1)for an-other48h in a final volume containing150μL medium;then, the medium were removed.After that,150μL of fresh medium and10μL of MTT(5g·L-1in PBS)were added to each well and the culture plates were incubated at37°C with5%CO2for 4h.After removal of the medium,150μL of DMSO was add-ed to each well to dissolve the dye.The absorbance at492nm was measured using a microplate reader.Each data point was derived from three parallel samples.3Results and discussionThe nanoscale graphene oxide(NGO)was synthesized by a modified Hummersʹmethod.Then the NGO was surface func-tionalized by attaching carboxylic acid and polyethylene glycol groups.Fig.1shows the schematic illustration of pegylation of graphene oxide.We have characterized the size of NGO-COOH-PEG using transmission electron microscopy.Fig.2shows the TEM image of NGO-COOH-PEG.One can see that the NGO-COOH-PEG (black dots)exists as platelet having lateral size less than40 nm.The structural evolution from NGO to NGO-COOH and to NGO-COOH-PEG was investigated using FTIR,the results are1521Acta Phys.⁃Chim.Sin .2012V ol.28shown in Fig.3.It can be seen that NGO has three evident ab-sorption bands at ~1053,1629,and 3411cm -1,which are from C ―O ―C ―,―C ＝O and ―OH stretching modes,respective-ly.20-22Besides when carboxylic acid group (―COOH)was connected to NGO to form NGO-COOH,the absorption at around 3430cm -1(from ―OH stretching mode)becomes weak.This is likely due to partial reduction of NGO so that some ―OH groups were consumed under strong alkaline con-dition.When PEG was attached further with the NGO-COOH to form NGO-COOH-PEG,the methylene (―CH 2―)stretch-ing vibration at 2850cm -1from the PEG-ylated reaction is more evident,consistent with the grafting of PEG molecules onto NGO-COOH sheets.The colloid solution zeta potential distributions were studied by measuring zeta potential.Fig.4shows the transformed zeta potential values of the synthesized NGO,NGO-COOH,and NGO-COOH-PEG in their water solution.It can be found thatthe potential distribution curve of NGO has a negatively charged peak with position centered at about -53.4mV ,indi-cating that the NGO water suspension can be stored stably.As for NGO-COOH,the peak position of its zeta potential shifts to -36.1mV .This means that the NGO-COOH water suspen-sion can also be stably stored.However,the peak position of the zeta potential for NGO-COOH-PEG shifts to -11.3mV .In-deed,the NGO-COOH-PEG solution is less stable compared to NGO solution,and NGO-COOH-PEG will precipitate from the water after a longer time store.For a material being used in biomedical area,its biocompati-bility and toxicology must be evaluated.Here,the NGO-COOH-PEG was suspended in water,phosphate buff-ered saline (PBS),and cell culture medium,respectively.We have examined the dispersive abilities of NGO and NGO-COOH-PEG in these three solutions.The results are shown in Fig.5.The experiment was performed at room tem-perature.Fig.5A shows the dispersion status of NGO in water,PBS,and cell culture medium,respectively.We find that NGO is able to uniformly disperse in water to form homogenous and stable solution for several days,but can not dispersed well in PBS and cell culture medium.However,NGO-COOH-PEG could be uniformly dispersed not only in water,but also in PBS and cell culture medium as shown in Fig.5B.The NGO-COOH-PEG could be stably dispersed in PBS and cell culture medium for several days.This is likely due to the screening of electrostatic charge and nonspecific binding of proteins on the NGO-COOH-PEG.14The use of nanomaterials in vitro and in vivo requires that they are biocompatible.23-27The cytotoxic behavior of NGO-COOH-PEG nanomaterials on cells was examined by the MTT assay.A549cell (lung adenocarcinoma cells)viability was as-sessed after exposure to different concentrations of NGO-COOH-PEG for 48h.Control experiments were conducted in a similar manner without the presence of the NGO-COOH-Fig.1Schematic illustration of PEG decoratedNGO-COOHFig.2TEM image ofNGO-COOH-PEGFig.3FTIR spectra of NGO (a),NGO-COOH (b),andNGO-COOH-PEG (c)Fig.4Zeta potential of NGO (A),NGO-COOH (B),andNGO-COOH-PEG(C)1522ZHANG Xiao et al .:Cell Biocompatibility of Functionalized Graphene OxideNo.6PEG.As shown in Fig.6,cell viability data indicated that the NGO-COOH-PEG did not significantly affect A549cell prolif-eration up to the concentration of 102.0mg ·L -1.The results showed that the NGO-COOH-PEG was reasonably nontoxic and biocompatible up to the given concentrations.The similar results were also found by other groups.For example,Zhang et al .14covalently conjugated graphene oxide with dextran (DEX)and incubated it with Hela cells.Their cellular experiments showed that DEX coating on GO offers remarkably reduced cell toxicity.However,our PEG-modified GO showed more uniform size which would be helpful for the next step experi-ments.4ConclusionsWe have adopted a modified Hummers ʹmethod to obtain graphite oxide and then dispersed them in ultra-pure water to form graphite oxide suspension.After multiple sonication and centrifugal screening,water-soluble NGO were received.The NGO sheets were surface functionalized by attaching ―COOH and PEG groups to form NGO-COOH-PEG struc-ture,which is well soluble in PBS and cell culture medium without agglomeration.Cell viability studies show that the functionalized NGO sheets have good solubility.Owing to itssmall size,large 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