Which material properties / features determine 

the biological response to carbon nanotubes? 

Robert Hurt 

Brown University, Providence Rhode Island 

Presented at NIST March 1st, 2011 
Examples from literature on 

carbon nanotube health risks 

- producing asbestos-like pathology in mice 

 [Poland et al.] 

- promising biocompatible materials for drug delivery 

 ....with no toxic side effects [Dai and coworkers] 

- showing only “false” toxicity through an adsorptive artifact 

 in a common toxicity assay [Worle-Knirsch et al.] 

- causing oxidative damage to mouse lung 

 and heart tissue [Lie et al.]

- toxic only if not purified; copepods [Templeton et al.] ; human lung cells [Krug et al.] 

- only toxic if not functionalized [Sayes et al.] 

The largest discrepancy is between 

 biomedical studies and toxicological studies 
Paracelsus:*1493/1541*

“father*of*toxicology”*

“All$things$are$toxic....”$

or$“the$dose$makes$the$poison”$

[$only$by$defining$and$limi<ng$dose$$$

can$something$be$regarded$as$$

non=toxic,$in$that$situa<on$]$

Toxics$affects$have$been$documented$for$

$ethanol,$vitamin$E,$oxygen,$water......$

Scien<fic$papers$on$nano=toxicity$will,$$

by$design,$report$and$discuss$toxic$effects$

Some%Basics.....%

Dose 

Biological 

response 

LD50, EC50, IC50 

Risk = Hazard x Exposure 
Simple Experiment 

SWNT removal by 

centrifugal ultrafiltration 

culture cells 

in “exposed” media 

SWNTs + Cell culture medium 

Viability of HepG2 liver cells 

Effect of SWNTs on Liver Cells 
Nanotubes Inhibit Cell Proliferation by Hydrophobic Adsorption of Folic Acid 

[Guo et al., Small, 4 (6) 721–727 2008] 

DNA synthesis 

Hydrophobic surfaces can also adsorb molecular 

probe dyes to give false positives in toxicity assays 

[Worle-Knirsch et al., 2006] 

Essential micronutrient 

 (vitamin) profiling 

after SWNT exposure 
“Trojan horse” 

mechanism 

For metal nanoparticles, 

H+-mediated corrosion at lysosomal

pH enhances ion release 

[Liu et al. “Bioavailability of Nickel in 

Single-Wall Carbon Nanotubes”, 

Advanced Materials, 2007] 

Metal Catalyst Residues 

in Carbon Nanotubes 
CNT iron catalyzes reactive oxygen species (ROS) 

generation and inflammatory reactions 

DNA damage 

Single-strand breaks 

in plasmid DNA model 

Studies on Fe effects in CNT biological response: 

Kagan et al., 2006; Guo et al., Chemistry of Materials, 2007; Koyama et al. Carbon, 2009 
Cell 

membrane 

Selectivity filter 

in neuronal voltage-gated 

calcium-ion channel 

Jakubek , Marangoudakis, Raingo, 

Liu, Lipscombe, Hurt, “The inhibition 

of neuronal calcium-ion channels by 

trace levels of yttrium released from 

carbon nanotubes,” Biomaterials, 

2009. 

Blocking of calcium ion 

channels in neurons by 

SWNTs is really due to 

release of trace 

quantities (<< 1 ppm) 

 of yttrium 
Leaching, or ion-particle partitioning, 

Is a major theme in nanomaterial safety 

Quantum dots 

Carbon nanotubes 

ZnO Ni 

NiO 

Antibacterial 

nanosilver 

Fe2+, Ni2+, Y3+, Co3+.... 

Cd2+ , Se2- , Zn2+ 

Zn2+ 

Ni2+ 
nAg 

Ag+ 

incubation 

cellulose membrane 

3K Da, (1-2 nm pore size) 

ultrafiltration 

separation 

Ag+ 

nAg 

total ionic 

silver by AAs 

(graphite 

furnace atomic 

absorption 

spectroscopy) 

or ICP 

quantification 

A general experimental method 

 to measure metal bioavailability 

or ion-nanoparticle partitioning 

[Liu et al. Env. Sci. Tech., 44:6 2169–2175 2010] 

Basic Experiment
Biological surface reactivity of CNTs 

Glutathione (GSH): 

the key intracellular antioxidant: 

Dr. Xinyuan Liu 

Dissolved O2 consumption Annealing 
35 nm 

diameter MWNTs 

glassy carbon 

carbon black 

SWNTs 

140 nm 

diameter MWNTs 

Biological surface reactivity 

O2#

.

O2

%

 -> other ROS -> GSH depletion

e CNTs oxidize through reduction ! - 

O2#

(

.

O2

%

)ads

GSH 

GSSG 

.

O2

%

2H+#+#.O2

% O2#+#H2O2#

GSH 

GSSG 

GSH 

GSSG 

Proposed 

mechanism 

eReactivity of other 

“graphenic” carbons 
The Fiber Pathogenicity Paradigm 

Asbestos-like mechanisms 

may arise for fibers with : 

d <~ 3 um 

(for inhalation into the deep lung) 

and 

 L > 10-20 um 

(for impaired clearance by macrophages) 

and 

biopersistence

(for long-term effects)

Asbestos 

fiber 

Mesothelium

(linings of lung and 

abdominal cavity) 

The Effect of CNT Geometry 

The Carbon Nanotube / Asbestos Analogy 

A. Kane and R. Hurt, Nature Nanotechnology, 2008

Prof. A. Kane 

Pathology and Laboratory Medicine 
“Carbon#nanotubes#introduced#

into#the#abdominal#cavity##

of#mice#show#asbestoslike#

pathogenicity#in#a#pilot#study”,##

Poland#et#al.,#Nature*

Nanotechnology,#2008#

There is evidence that long length (> 10-20 um) 

impairs lung clearance and also causes cytotoxicity 

asbestos 

MWNTs 

MWNTs 
Carboxylate-functionalized SWNTs do degrade 

during 90 days in phagolysosomal simulant fluid 

Biopersistence / biodegradability

- Biopersistence is the single most important material 

 property influencing toxicity of respirable fibers. 

- Carbon does not dissolve, but can, 

 in principle, be oxidized 

- Environmental persistence greatly increases the 

 potential adverse impact of a toxicant 
Sample Functionalization Observation

SWNTs

(d:1-2nm)

Non-functionalized

Aryl sulfonate

Ozonate

1000oC treated

Carboxylate

Commercial (nitric acid treated)

In-house functionalized 

(mixed acid treated) 

 15mi 

 1hr

$$$$3hr 

No significant change

No significant change

No significant change

No significant change

No significant change

Visible degradation

No significant change

Visible degradation

Visible degradation

MWNTs

(d: 35±10nm)

Non-functionalized

Aryl-sulfonate

No significant change

No significant change

Reference 

Materials

CNFs (d: 200nm)

Non-functionalized

Aryl sulfonate

Wollasonite

Crocidite Asbestos (d: 30-150nm)

No significant change

No significant change

Dissolved

No significant change 

Only a few nanotube types show 

visible biodegradation 

 [Liu et al., ”Biodurability....”, Carbon, 2010]

The only degradable nanotubes 

are SWNTs treated with oxidizing acids 

(HNO3 / H2SO4), which are known to 

produce COOH groups 
A hypothesis for carbon nanotubes: 

Toxicity / biocompatibility is determined by multiple material features 

Toxicity Biocompatibility 

Deeper purification 

(removal of metals, amorphous carbon) 

Progressive length reduction 

(through cutting / sorting) 

Increasing surface 

functionalization for hydrophilicity 

CNTs are a good case study in that material features and formulation appear to matter 

Reduced tip availability (CNT substrates) 

Biodegradability? 
What can we do with this information? 

Case I: Engineered CNTs in end applications 

 Data suggest real opportunities to design for safety, through: 

- Binding to substrates or in matrices (no free tips) 

- Shortening 

- Deep purification 

- Surface functionalization for hydrophilicity 

- Biodegradability (?) 

 Can/should we define different hazard categories? 

Case II: Primary manufacture 

 Exposures likely occur before opportunity to engineer properties 

 So: control exposure 

 - process release / respirators / air filtration

Case III: Misuse, Accident Scenarios, and End-of-Life 

- Manufacturers lose control of their products after sale 

- We need to envision, assess, and manage risk is multiple scenarios 

 by managing hazard or exposure 
Contributors 

Materials Chemistry Pathobiology 

Prof. Robert Hurt Prof. Agnes Kane 

Xinyuan Liu Prof. Charles A. Vaslet 

Lorin Jakubek Ashley Smith 

Aihui Yan Dr. Annette Von Dem Bussche 

Lin Guo Kevin McNeil

Dr. Indrek Kulaots Vanesa Sanchez 

Daniel Morris Jody Pietruska 

 

Neuroscience 

Prof. Diane Lipscombe 

Jessica Raingo 

Spiro Marangoudakis

Financial support is acknowledged from the NIEHS R01 and Superfund basic 

research program, NSF NIRT program, EPA STAR grants 

RD-83171901-0