Biocompatibility of pristine graphene for neuronal interface

Laboratory investigation

View More View Less
  • 1 Department of Neurosurgery, Baylor College of Medicine;
  • | 2 Division of Pediatric Neurosurgery, Texas Children's Hospital;
  • | 3 Smalley Institute for Nanoscale Science and Technology, Chemistry Department, Rice University;
  • | 5 Interdepartmental Program in Translational Biology and Molecular Medicine, Departments of Neurosurgery and Neurology, Baylor College of Medicine;
  • | 6 Michael E. DeBakey VA Medical Center, Houston, Texas; and
  • | 4 College of Environment and Biotechnology, Chongqing Technology and Business University, Chongqing, People's Republic of China
Restricted access

Purchase Now

USD  $45.00

JNS + Pediatrics - 1 year subscription bundle (Individuals Only)

USD  $515.00

JNS + Pediatrics + Spine - 1 year subscription bundle (Individuals Only)

USD  $612.00
Print or Print + Online

Object

Graphene possesses unique electrical, physical, and chemical properties that may offer significant potential as a bioscaffold for neuronal regeneration after spinal cord injury. The purpose of this investigation was to establish the in vitro biocompatibility of pristine graphene for interface with primary rat cortical neurons.

Methods

Graphene films were prepared by chemical vapor deposition on a copper foil catalytic substrate and subsequent apposition on bare Permanox plastic polymer dishes. Rat neuronal cell culture was grown on graphene-coated surfaces, and cell growth and attachment were compared with those on uncoated and poly-d-lysine (PDL)-coated controls; the latter surface is highly favorable for neuronal attachment and growth. Live/dead cell analysis was conducted with flow cytometry using ethidium homodimer-1 and calcein AM dyes. Lactate dehydrogenase (LDH) levels—indicative of cytotoxicity—were measured as markers of cell death. Phase contrast microscopy of active cell culture was conducted to assess neuronal attachment and morphology.

Results

Statistically significant differences in the percentage of live or dead neurons were noted between graphene and PDL surfaces, as well as between the PDL-coated and bare surfaces, but there was little difference in cell viability between graphene-coated and bare surfaces. There were significantly lower LDH levels in the graphene-coated samples compared with the uncoated ones, indicating that graphene was not more cytotoxic than the bare control surface. According to phase contrast microscopy, neurons attached to the graphene-coated surface and were able to elaborate long, neuritic processes suggestive of normal neuronal metabolism and morphology.

Conclusions

Further use of graphene as a bioscaffold will require surface modification that enhances hydrophilicity to increase cellular attachment and growth. Graphene is a nanomaterial that is biocompatible with neurons and may have significant biomedical applications.

Abbreviations used in this paper:

CNT = carbon nanotubes; CVD = chemical vapor deposition; EthD-1 = ethidium homodimer-1; FACS = fluorescence-activated cell sorting; GO = graphene oxide; LDH = lactate dehydrogenase; PDL = poly-d-lysine; PMMA = polymethylmethacrylate; SCI = spinal cord injury; TEM = transmission electron microscopy; XPS = x-ray photoelectron spectroscopy.

JNS + Pediatrics - 1 year subscription bundle (Individuals Only)

USD  $515.00

JNS + Pediatrics + Spine - 1 year subscription bundle (Individuals Only)

USD  $612.00
  • 1

    Agarwal S, , Zhou X, , Ye F, , He Q, , Chen GC, & Soo J, et al.: Interfacing live cells with nanocarbon substrates. Langmuir 26:22442247, 2010

  • 2

    Akhavan O, & Ghaderi E: Toxicity of graphene and graphene oxide nanowalls against bacteria. ACS Nano 4:57315736, 2010

  • 3

    Allen MJ, , Tung VC, & Kaner RB: Honeycomb carbon: a review of graphene. Chem Rev 110:132145, 2010

  • 4

    Balandin AA, , Ghosh S, , Bao W, , Calizo I, , Teweldebrhan D, & Miao F, et al.: Superior thermal conductivity of single-layer graphene. Nano Lett 8:902907, 2008

    • Search Google Scholar
    • Export Citation
  • 5

    Basko DM: Calculation of the Raman G peak intensity in monolayer graphene: role of Ward identities. New J Phys 11:095011, 2009

  • 6

    Belyanskaya L, , Weigel S, , Hirsch C, , Tobler U, , Krug HF, & Wick P: Effects of carbon nanotubes on primary neurons and glial cells. Neurotoxicology 30:702711, 2009

    • Search Google Scholar
    • Export Citation
  • 7

    Busch SA, , Horn KP, , Silver DJ, & Silver J: Overcoming macrophage-mediated axonal dieback following CNS injury. J Neurosci 29:99679976, 2009

    • Search Google Scholar
    • Export Citation
  • 8

    Castro EV, , Novoselov KS, , Morozov SV, , Peres NM, , dos Santos JM, & Nilsson J, et al.: Biased bilayer graphene: semiconductor with a gap tunable by the electric field effect. Phys Rev Lett 99:216802, 2007

    • Search Google Scholar
    • Export Citation
  • 9

    Chang Y, , Yang ST, , Liu JH, , Dong E, , Wang Y, & Cao A, et al.: In vitro toxicity evaluation of graphene oxide on A549 cells. Toxicol Lett 200:201210, 2011

    • Search Google Scholar
    • Export Citation
  • 10

    Chen H, , Müller MB, , Gilmore KJ, , Wallace GG, & Li D: Mechanically strong, electrically conductive, and biocompatible graphene paper. Adv Mater 20:35573561, 2008

    • Search Google Scholar
    • Export Citation
  • 11

    De Arco LG, , Yi Z, , Kumar A, & Chongwu Z: Synthesis, transfer, and devices of single- and few-layer graphene by chemical vapor deposition. IEEE Trans Nanotechnol 8:135138, 2009

    • Search Google Scholar
    • Export Citation
  • 12

    Dimiev A, , Kosynkin DV, , Sinitskii A, , Slesarev A, , Sun Z, & Tour JM: Layer-by-layer removal of graphene for device patterning. Science 331:11681172, 2011

    • Search Google Scholar
    • Export Citation
  • 13

    Gall NR, , Rut'kov EV, & Tontegode AY: Two dimensional graphite films on metals and their intercalation. Int J Mod Phys B 11:18651911, 1997

    • Search Google Scholar
    • Export Citation
  • 14

    Geim AK: Graphene: status and prospects. Science 324:15301534, 2009

  • 15

    Geim AK, & Novoselov KS: The rise of graphene. Nat Mater 6:183191, 2007

  • 16

    Gupta A, , Chen G, , Joshi P, , Tadigadapa S, & Eklund PC: Raman scattering from high-frequency phonons in supported n-graphene layer films. Nano Lett 6:26672673, 2006

    • Search Google Scholar
    • Export Citation
  • 17

    Han MY, , Ozyilmaz B, , Zhang Y, & Kim P: Energy band-gap engineering of graphene nanoribbons. Phys Rev Lett 98:206805, 2007

  • 18

    Heo C, , Yoo J, , Lee S, , Jo A, , Jung S, & Yoo H, et al.: The control of neural cell-to-cell interactions through non-contact electrical field stimulation using graphene electrodes. Biomaterials 32:1927, 2011

    • Search Google Scholar
    • Export Citation
  • 19

    Hu W, , Peng C, , Luo W, , Lv M, , Li X, & Li D, et al.: Graphene-based antibacterial paper. ACS Nano 4:43174323, 2010

  • 20

    Jia X, , Hofmann M, , Meunier V, , Sumpter BG, , Campos-Delgado J, & Romo-Herrera JM, et al.: Controlled formation of sharp zigzag and armchair edges in graphitic nanoribbons. Science 323:17011705, 2009

    • Search Google Scholar
    • Export Citation
  • 21

    Jin Z, , Lomeda JR, , Price BK, , Lu W, , Zhu Y, & Tour JM: Mechanically assisted exfoliation and functionalization of thermally converted graphene sheets. Chem Mater 21:30453047, 2009

    • Search Google Scholar
    • Export Citation
  • 22

    Kalbacova M, , Broz A, , Kong J, & Kalbac M: Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells. Carbon 48:43234329, 2010

    • Search Google Scholar
    • Export Citation
  • 23

    Kim KS, , Zhao Y, , Jang H, , Lee SY, , Kim JM, & Kim KS, et al.: Large-scale pattern growth of graphene films for stretchable transparent electrodes. Nature 457:706710, 2009

    • Search Google Scholar
    • Export Citation
  • 24

    Korzeniewski C, & Callewaert DM: An enzyme-release assay for natural cytotoxicity. J Immunol Methods 64:313320, 1983

  • 25

    Li X, , Cai W, , An J, , Kim S, , Nah J, & Yang D, et al.: Large-area synthesis of high-quality and uniform graphene films on copper foils. Science 324:13121314, 2009

    • Search Google Scholar
    • Export Citation
  • 26

    Liao KH, , Lin YS, , Macosko CW, & Haynes CL: Cytotoxicity of graphene oxide and graphene in human erythrocytes and skin fibroblasts. ACS Appl Mater Interfaces 3:26072615, 2011

    • Search Google Scholar
    • Export Citation
  • 27

    Liu Z, , Robinson JT, , Sun X, & Dai H: PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. J Am Chem Soc 130:1087610877, 2008

    • Search Google Scholar
    • Export Citation
  • 28

    Mattevi C, , Kim H, & Chhowalla M: A review of chemical vapour deposition of graphene on copper. J Mater Chem 21:33243334, 2011

  • 29

    Myung S, , Solanki A, , Kim C, , Park J, , Kim KS, & Lee KB: Graphene-encapsulated nanoparticle-based biosensor for the selective detection of cancer biomarkers. Adv Mater 23:22212225, 2011

    • Search Google Scholar
    • Export Citation
  • 30

    Novoselov KS, , Geim AK, , Morozov SV, , Jiang D, , Katsnelson MI, & Grigorieva IV, et al.: Two-dimensional gas of massless Dirac fermions in graphene. Nature 438:197200, 2005

    • Search Google Scholar
    • Export Citation
  • 31

    Novoselov KS, , Geim AK, , Morozov SV, , Jiang D, , Zhang Y, & Dubonos SV, et al.: Electric field effect in atomically thin carbon films. Science 306:666669, 2004

    • Search Google Scholar
    • Export Citation
  • 32

    Rajnicek AM, , Robinson KR, & McCaig CD: The direction of neurite growth in a weak DC electric field depends on the substratum: contributions of adhesivity and net surface charge. Dev Biol 203:412423, 1998

    • Search Google Scholar
    • Export Citation
  • 33

    Reina A, , Jia X, , Ho J, , Nezich D, , Son H, & Bulovic V, et al.: Large area, few-layer graphene films on arbitrary substrates by chemical vapor deposition. Nano Lett 9:3035, 2009

    • Search Google Scholar
    • Export Citation
  • 34

    Reina A, , Thiele S, , Jia X, , Bhaviripudi S, , Dresselhaus M, & Schaefer J, et al.: Growth of large-area single- and bi-layer graphene by controlled carbon precipitation on polycrystalline Ni surfaces. Nano Res 2:509516, 2009

    • Search Google Scholar
    • Export Citation
  • 35

    Ryoo SR, , Kim YK, , Kim MH, & Min DH: Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies. ACS Nano 4:65876598, 2010

    • Search Google Scholar
    • Export Citation
  • 36

    Sanchez VC, , Pietruska JR, , Miselis NR, , Hurt RH, & Kane AB: Biopersistence and potential adverse health impacts of fibrous nanomaterials: what have we learned from asbestos?. Wiley Interdiscip Rev Nanomed Nanobiotechnol 1:511529, 2009

    • Search Google Scholar
    • Export Citation
  • 37

    Sasidharan A, , Panchakarla LS, , Chandran P, , Menon D, , Nair S, & Rao CN, et al.: Differential nano-bio interactions and toxicity effects of pristine versus functionalized graphene. Nanoscale 3:24612464, 2011

    • Search Google Scholar
    • Export Citation
  • 38

    Shapiro S, , Borgens R, , Pascuzzi R, , Roos K, , Groff M, & Purvines S, et al.: Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial. J Neurosurg Spine 2:310, 2005

    • Search Google Scholar
    • Export Citation
  • 39

    Sisken BF, , Walker J, & Orgel M: Prospects on clinical applications of electrical stimulation for nerve regeneration. J Cell Biochem 51:404409, 1993

    • Search Google Scholar
    • Export Citation
  • 40

    Stankovich S, , Dikin DA, , Dommett GH, , Kohlhaas KM, , Zimney EJ, & Stach EA, et al.: Graphene-based composite materials. Nature 442:282286, 2006

  • 41

    Sucapane A, , Cellot G, , Prato M, , Giugliano M, , Parpura V, & Ballerini L: Interactions between cultured neurons and carbon nanotubes: a nanoneuroscience vignette. J Nanoneurosci 1:1016, 2009

    • Search Google Scholar
    • Export Citation
  • 42

    Sun Z, , Kohama S, , Zhang Z, , Lomeda J, & Tour J: Soluble graphene through edge-selective functionalization. Nano Res 3:117125, 2010

  • 43

    Sun Z, , Yan Z, , Yao J, , Beitler E, , Zhu Y, & Tour JM: Growth of graphene from solid carbon sources. Nature 468:549552, 2010

  • 44

    Wang J, , Sun P, , Bao Y, , Liu J, & An L: Cytotoxicity of single-walled carbon nanotubes on PC12 cells. Toxicol In Vitro 25:242250, 2011

  • 45

    Wang K, , Ruan J, , Song H, , Zhang J, , Wo Y, & Guo S, et al.: Biocompatibility of graphene oxide. Nanoscale Res Lett 6:8, 2011

  • 46

    Wang Y, , Li Z, , Wang J, , Li J, & Lin Y: Graphene and graphene oxide: biofunctionalization and applications in biotechnology. Trends Biotechnol 29:205212, 2011

    • Search Google Scholar
    • Export Citation
  • 47

    Watcharotone S, , Dikin DA, , Stankovich S, , Piner R, , Jung I, & Dommett GH, et al.: Graphene-silica composite thin films as transparent conductors. Nano Lett 7:18881892, 2007

    • Search Google Scholar
    • Export Citation
  • 48

    Wu C, , Zhou Y, , Miao X, & Ling L: A novel fluorescent biosensor for sequence-specific recognition of double-stranded DNA with the platform of graphene oxide. Analyst (Lond) 136:21062110, 2011

    • Search Google Scholar
    • Export Citation
  • 49

    Yang W, , Ratinac KR, , Ringer SP, , Thordarson P, , Gooding JJ, & Braet F: Carbon nanomaterials in biosensors: should you use nanotubes or graphene?. Angew Chem Int Ed Engl 49:21142138, 2010

    • Search Google Scholar
    • Export Citation
  • 50

    Yao L, , Shanley L, , McCaig C, & Zhao M: Small applied electric fields guide migration of hippocampal neurons. J Cell Physiol 216:527535, 2008

    • Search Google Scholar
    • Export Citation
  • 51

    Zhang Y, , Ali SF, , Dervishi E, , Xu Y, , Li Z, & Casciano D, et al.: Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. ACS Nano 4:31813186, 2010

    • Search Google Scholar
    • Export Citation

Metrics

All Time Past Year Past 30 Days
Abstract Views 1208 375 29
Full Text Views 213 22 0
PDF Downloads 201 24 0
EPUB Downloads 0 0 0