presented at
SNO in Southern California, November, 2013
Graphene Microsheets Enter Cells
through Spontaneous Membrane Penetration
at Edge Asperities and Corner Sites*
Yinfeng Li, Hongyan Yuan, Annette von dem Bussche, Megan Creighton,
Robert H. Hurt, Agnes B. Kane and Huajian Gao
School of Engineering, Department of Pathology and Laboratory Medicine,
Institute for Molecular and Nanoscale Innovation
Brown University, Providence, RI, 02912, USA.
Department of Engineering Mechanics, Shanghai Jiao Tong University,
Shanghai 200240, China
* Based on Li et al., Proc. Nat. Academy of Sci., 110 (30) 12295–12300 (2013)
We now have a family of two-dimensional graphene-based materials
few-layer graphene;
multilayer graphene;
graphite nanoplates*
graphene
oxide
Natural
flake graphite
Graphite bisulfate
or
Graphite oxide
Expanded graphite
H2SO4 (intercalant)
oxidizing agent(s)Rapid thermal
annealing
Wet sonication
Graphene – calling all researchers in sustainable nanotechnology
benzene, naphthalene, phenanthrene, higher PAH,...”graph”-ene
------------------------------- molecular weight --------------------------> [Boehm et al., Carbon, 1986]
* “All in the graphene family – A recommended
nomenclature for two-dimensional carbon materials”, Carbon, 2013
isolated in 2004
Some emerging applications of graphene materials
Materials with engineered folds and wrinkles
“Ruga materials” actuators, crumpled particles, filled sacks
Ultrathin coatings as molecular barriers
Catalyst supports - ultrahigh surface area (2600/N m2/g)
Emulsion stabilizers with 100% atom efficiency
Low-percolation-threshold composite fillers
(for e-conductivity, strength, barrier properties)
Electrode materials (e-conductivity, intercalation)
Thin conductive films; conducting inks
Guo et al.,
ES&T, 2012
Biological interactions and safety of graphene materials
Sanchez, Jachak, Hurt, Kane, “Biological Interactions of Graphene-Family Nanomaterials
– An Interdisciplinary Review” Chemical Research in Toxicology, 25 (1) 15–34 (2012).
Unique modes of biological coupling for atomically thin plates
Graphene Materials Can Produce Artifacts during In Vitro Toxicity Testing
Creighton, Hurt, Kane et al., “Graphene-Induced
Optical and Adsorptive Artifacts During In Vitro
Toxicology Assays” Small, 2013
GFNs deplete folic acid
from cell culture medium
GFNs adsorb and quench
dyes used in toxicity assays
Are Graphene-Based Powders an Inhalation Health Risk?
For commercial
multi-layer graphene
samples:
A = 2600/N
(m2/g)
Comparisons to carbon nanotubes
Differences
- graphene materials have fewer impurities;
- lie outside the fiber pathogenicity paradigm;
- are atomically thin
Similarities
- graphene materials have range of
geometries within the family
> thickness range: 0.34 – 30 nm
> lateral dimension range: 5 nm – 100 um
(factor of 20,000 !)
- have range of surface chemistries
Deposition patterns
for monolayer graphene
in human respiratory tract
Can graphene materials penetrate
cell membranes and be internalized?
Huajian Gao Agnes Kane Robert Hurt
MWNT entry
into liver cells
Shi, Von dem Bussche, Hurt, Kane, Gao
“Cell entry of one-dimensional nanomaterials
occurs by tip recognition and rotation
Nature Nanotechnology, 2011
Prior work on carbon nanotube uptake
2 um
Example Morphologies in Commercial Multi-Layer Graphene
Few-layer graphene microsheets enter cells
and localize in the cytoplasm or in vesicles
human lung
epithelial cells
mouse
macrophages
all FLG microsheets are internalized
sheets localize parallel to substrate
larger sheets alter cytoskeletal structure
human lung
epithelial cells
mouse
macrophages
primary human
keratinocytes
High-resolution imaging of the cell entry process
Coarse-Grained and All-Atom Molecular Dynamics Simulations(H. Gao group)
Coarse-grained simulations:
POPC lipid bilayer with 6.4 nm
lateral dimension graphene nanosheet
All-atom simulations:
POPC lipid bialyer interacting with
monolayer graphene sheet corners
These simulations are either:
Spontaneous (entry initiated by thermal fluctuations)
or
“Steered” – pulled through membrane by virtual spring to calculate energy barriers
Molecular Dynamics Results: Graphene NanoSheets Penetrate Cell Membranes
but Graphene MicroSheets do not….?
calculation of
energy barriers
for penetration
(all-atom MD)
Microsheets are repelled
away by entropic forces,
even during edge-on approach
Nanosheet
spontaneous entry
(CGMD)
only ∼5kBT
energy barrier
Y Li, H Yuan, A von dem Bussche, M Creighton, RH Hurt, AB Kane, and H Gao,
“Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites”,
Proceedings of the National Academy of Sciences, 2013.
Resolution: “Real” graphene samples do not have atomically smooth edges!
Other irregular edge geometries
Asperities and corners initiate passive penetration,
which propagates along the graphene edge
Reference….
[Sanchez, Jachack, Hurt, Kane, Chemical Research in Toxicology, 2012]
500 nm 800 nm 5 um
25 um
Uptake
Attachment and
multicellular coverage
Graphene lateral size determines
macrophage uptake and lung clearance
The Hurt
Laboratory
at Brown
Financial support was provided by NSF CBET-
1132446 (Barbara Karn, program manager),
and NSF CMMI-1028530
and the NIEHS Superfund Research Program
Summary Statement
Graphene-based materials are a new material family with varying geometry and chemistry.
Materials with lateral dimension < about 5 um can enter mammalian cells initiated by
spontaneous penetration of lipid bilayers at atomically-thin corner and rough edge sites.
Uptake/ clearance for large lateral dimension (> 5 um) flakes is often incomplete, and this subset
of materials may deserve special attention in nanotoxicity studies and risk assessment
Backups
Large GFNs and Carbon Nanotubes Induce Macrophage Toxicity
2. Ultrathin Barrier Coatings
[Guo, Hurt et al., “Graphene-based environmental
barriers”, Envir. Sci. Tech. 2012]
With GO coating
See also: [V. Berry, “Impermeability of graphene
and its applications (Review), Carbon, in press 2013]
50 μm
1
10.5
comp
polym
P
P
GO Film Thickness (nm)
Poisson
disk
deposition
Polymer
4. Interfacial assembly: high-performance
emulsion stabilizers
200 um
200 um
50 ppm
GO
250 ppm
GO
GO SDS
Graphene oxide
structure
[Cote et al. Pure Appl.
Chem. 2011]
Unique features of graphene-based stabilizers
- Up to 100% atom economy
- conformal coverage
- multilayer tiling
- barrier properties?“Pickering emulsions”
Meg Creighton
Thermodynamic modelling
-ΔGstabilization = solid-oil – solid-water – ϒoil-water
oil-in-water emulsions
Examples of unique features of graphene-based stabilizers
1 221
1
2
50 um
Atomically thin GO sheets show
wetting transparency [Koratkar et al]
Atomically thin GO sheets crumple if oil phase evaporates
2. vdW transparency
3. Templating of crumpled microparticles
atomically
thin plates
10 nm spheres
Stabilization
Energy
(normalized)
1. Mass potency
21
200 nm
Nanodroplet Activated and Guided
Folding of Graphene Nanostructures[Patra, Wang, Kral, Nano Letters, 2009]
5. Folded structures – “Ruga” materials
Critical length for
graphene self-folding = (C/ )1/2
[Cranford, Sen, Buehler, 2009]
C – bending stiffness
- adhesion energy
Multilayers: bending stiffness ~ N3
(beam theory, no sliding)
K.S. Kim
Brown Univ
Ruga (pl Rugea) from Latin – folds, creases, wrinkles
Ruga materials are a new class of engineered structures
made by contraction of elastic substrates with stiffer surface films
citrate-stabilized
nanosilver as minority
phase (Ag:GO 1:16)citrate-stabilized nanosilver
as majority phase (Ag:GO 2:1)
Cargo-Filled Graphene NanosacksChen Y, Guo F, Jachak A, Kim S-P, Datta D, Liu J, Kulaots I, Vaslet C,
Jang HD, Huang J, Kane A, Shenoy VB, Hurt RH, Nano Letters (2012).
salmon-sperm
DNA
?
Filled Graphene Nanosacks as Multifunctional Materials[Y Chen, F Guo, Y Qiu, H Hu, I Kulaots, E Walsh, RH Hurt, ACS Nano, 2013]
Ternary hybrids as MRI / CT dual contrast agents
gold / iron oxide barium titanate / iron oxide Full scale, clinical MRI / CT results
gold nanoparticles silicon nanoparticles iron oxide NPs
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