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