Pharmacokinetics, metabolism and toxicity of carbon nanotubes for biomedical purposes.

Abstract

Carbon nanotubes (CNTs) have attracted great interest of the nano community and beyond. However, the biomedical applications of CNTs arouse serious concerns for their unknown in vivo consequence, in which the information of pharmacokinetics, metabolism and toxicity of CNTs is essential. In this review, we summarize the updated data of CNTs from the biomedical view. The information shows that surface chemistry is crucial in regulating the in vivo behaviors of CNTs. Among the functionalization methods, PEGylation is the most efficient one to improve the pharmacokinetics and biocompatibility of CNTs. The guiding effects of the pharmacokinetics, metabolism and toxicity information on the biomedical applications of CNTs are discussed.

Full text

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T
Th he er ra an no os st ti ic cs s
2012; 2(3):271-282. doi: 10.7150/thno.3618
Review
Pharmacokinetics, Metabolism and Toxicity of Carbon Nanotubes for Bio-
medical Purposes
Sheng-Tao Yang1,2, Jianbin Luo1, Qinghan Zhou1, Haifang Wang2,
1. College of Chemistry and Environment Protection Engineering, Southwest University for Nationalities, Chengdu 610041, China.
2. Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai 200444, China.
 Corresponding author: Prof. Haifang Wang, Institute of Nanochemistry and Nanobiology, Shanghai University, Shanghai
200444, China. Tel: 86-21-66135276; E-mail: hwang@shu.edu.cn
© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2011.10.10; Accepted: 2011.11.20; Published: 2012.03.05
Abstract
Carbon nanotubes (CNTs) have attracted great interest of the nano community and beyond.
However, the biomedical applications of CNTs arouse serious concerns for their unknown in
vivo consequence, in which the information of pharmacokinetics, metabolism and toxicity of
CNTs is essential. In this review, we summarize the updated data of CNTs from the bio-
medical view. The information shows that surface chemistry is crucial in regulating the in vivo
behaviors of CNTs. Among the functionalization methods, PEGylation is the most efficient
one to improve the pharmacokinetics and biocompatibility of CNTs. The guiding effects of the
pharmacokinetics, metabolism and toxicity information on the biomedical applications of
CNTs are discussed.
Key words: Carbon nanotubes, pharmacokinetics, metabolism, toxicity, biomedical applications.
Introduction
Since the discovery, carbon nanotubes (CNTs)
have attracted great interest for their potential appli-
cations in various areas [1]. In biomedical area, CNTs
have been widely used in bio-analysis, therapeutics,
drug delivery and early diagnostics. These applica-
tions are booming in recent years [2-4]. For example,
Moon et al. showed that CNTs could target and de-
struct tumors by photothermal effect in situ [5]. Liu et
al. reported that CNTs could deliver doxorubicin and
paclitaxel to tumors to enhance the tumor growth
inhibition [6, 7]. The applications of CNTs in Raman
imaging [8], near infrared (NIR) fluorescence imaging
[9] and radiological imaging [10] were also reported.
Moreover, there is a trend to combine the therapeutics
and early diagnostics (namely theranostics) together
[11-13]. Such combinations on CNTs have been done
[2, 14].
With the rapid development of CNTs in bio-
medical area, the safety concern is aroused and stud-
ied among scientists of chemistry, pharmacology and
toxicology [15, 16]. Our group documented that the
information on the absorption, distribution, metabo-
lism, excretion/toxicity (ADME/T) of CNTs was in-
dispensible for the safety studies [17]. We unveiled
that CNTs were easily trapped in reticuloendothelial
system (RES) and induced slight toxicity to liver and
lungs [18-20]. Other reports suggested that CNTs
were nontoxic after careful surface modification [21,
22]. However, the guiding
theranostics is still limited to date.
In this review, we summarize the updated data
of CNTs from the biomedical view with focus on the
pharmacokinetics, metabolism and toxicity of CNTs in
vivo. We intend to elucidate the connection between
the pharmacological applications and toxicological
information of CNTs. The importance of surface
information for
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chemistry in regulating the in vivo behavior of CNTs is
stressed.
Pharmacokinetics and biodistribution of
CNTs
The pharmacokinetics and biodistribution of
CNTs have been studied as long as the safety issue of
CNTs was proposed [23]. The blood circulation time
and accumulation of CNTs in animal affect their bio-
medical applications and toxicity. Serving as a drug
vehicle, the long blood-circulated CNTs may accu-
mulate more in tumor, making them efficient for the
cancer diagnostics and treatment [24]. To study the
pharmacokinetics and biodistribution of CNTs, vari-
ous analytical techniques have been developed for
measuring CNTs in bio-samples [17]. With more in-
formation collected, we might get a chance to know
how these data guide the biomedical applications of
CNTs.
Pristine CNTs are hydrophobic and easy to ag-
gregate. It is difficult to disperse them in biosystems.
Studying pristine CNTs is in the pure toxicological
evaluations as well as the professional risk assess-
ments. For the biomedical applications, CNTs are
dispersed with the aid of suspending reagents or
chemical functionalization. Here, we only discuss the
pharmacokinetics and biodistribution of these dis-
persible CNTs.
CNTs, which are simply dispersed with surfac-
tant, are easily cleared from blood circulation and
accumulate in RES organs. We studied the biodistri-
bution of Tween 80 dispersed single-walled CNTs
(SWCNTs) in mice (Figure 1) [19]. The skeleton of
SWCNTs was labeled with stable isotope 13C and the
SWCNT contents in tissues were acquired by meas-
uring the
SWCNTs were fully cleared from blood circulation at
13C abundance. Tween 80 dispersed
1 day post intravenous injection. They were easily
recognized by macrophages and thereby captured by
RES in liver and spleen. These SWCNTs remained in
liver and spleen for over 3 months [20]. The less dis-
persed SWCNTs forming larger aggregates were fil-
tered by pulmonary capillary vessels. According to
the results of 13C abundance measurements, CNT
content in lungs decreased in the first month postex-
posure. Very similar results were reported by
Cherukuri et al. They dispersed SWCNTs with a sur-
factant named Pluronic F108 and the sample was
centrifuged at 100,000 g for 4 hours to remove the
aggregated SWCNTs [25]. By measuring the charac-
teristic NIR fluorescence of individual SWCNTs, they
found that Pluronic F108 dispersed SWCNTs majorly
accumulated in liver, while the pulmonary uptake
was undetectable. Considering only individual
SWCNTs emit NIR fluorescence, it is unknown from
this case whether SWCNTs aggregate in vivo or not.
For multi-walled CNTs (MWCNTs), similar results
were reported. Tween 80 dispersed MWCNTs accu-
mulated in liver and spleen with a short blood circu-
lation time [26]. Serum dispersed MWCNTs accumu-
lated in lungs [27]. In a word, the simply dispersed
CNTs are easily recognized by macrophages and
cleared from blood very fast. This characteristic limits
their application in drug delivery. However, simply
dispersed CNTs may find their position in diagnostics
and thermal therapy. Yang et al. reported that CNTs
could be recognized by lymph cells and migrate along
lymph vessels [28]. Thus, CNTs may be promising
materials for sentinel lymph node detection, which is
crucial in cancer therapy. Moreover, the slow migra-
tion of CNTs makes them ideal for thermal therapy,
because CNTs injected in tumor would stay for a long
time. Under NIR irradiation or in radiofrequency
field, the heat generated by CNTs could kill tumor
cells [5, 29].

Figure 1. Biodistribution of Tween 80 dispersed SWCNTs
in mice after intravenous injection. Adapted from reference
[19] with permission.

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By carefully choosing suspending reagents, the
pharmacokinetics and biodistribution of CNTs can be
improved significantly. Dai and coworkers developed
polyethylene glycol-phospholipid (PEG-PL) dis-
persed CNTs for biomedical detection, imaging,
thermal therapy and drug delivery. PEG is one of the
most efficient reagents to improve the pharmacoki-
netics behavior of exotic substances by shielding the
exotic substances from the opsonin recognition [30].
PEG chains were linked to PL molecules to form a
hydrophilic-hydrophobic structure [24, 31]. PEG-PL
suspended CNTs undergo hydrophobic interaction
between PL and CNTs. PEG-PL prolonged the blood
circulation half-life and reduced the RES capture of
SWCNTs in vivo. Liu
PEG2000-PL/SWCNTs (PEG-PL dispersed SWCNTs;
the molecule weight of PEG: 2000) had a half-life of
1.2 hours [24]. The half-life could be further prolonged
by using longer PEG chains and even branched PEG
chains. The circulation
PEG5000-PL/SWCNTs increased to about 5 hours [31].
Using branched PEG chains, the value could be higher
than 15 hours. More recently, Prencipe et al. used

et al. reported that
half-life of
γPGA-Py-mPEG
id)-pyrine(30%)-poly (ethylene glycol) methyl ethers)
(70%)] to disperse SWCNTs and studied their blood
circulation half-life [32]. The dispersion was very sta-
ble and prolonged the circulation half-life of SWCNTs
to 22.1 hours. Accordingly, the RES capture decreased
along with the change of PEG-PL molecules. The he-
patic uptake of PEG2000-PL/SWCNTs was 70%ID/g
(% injected dose per gram tissue), which decreased to
40%ID/g for PEG5000-PL/SWCNTs and 30%ID/g for
br-PEG7000-PL/SWCNTs (br: branched). The PEG
density on SWCNTs also regulated the blood circula-
tion, biodistribution and tumor uptake of SWCNTs.
The blood circulation half-life increased along with
increasing the PEG density, while the RES uptake
decreased accordingly [33]. The good behaviors of
PEG-PL/SWCNTs in vivo make them very useful in
biomedical areas. PEG-PL/SWCNTs have been used
to target tumors and deliver doxorubicin and
paclitaxel for cancer therapy [6, 7]. They have also
been used for photothermal ablation of tumors [33]. In
addition, they have been applied in Raman and pho-
toacoustic imaging for diagnostics [10, 34].
[poly-(γ-glutamic ac-

Figure 2. Pharmacokinetics of PEG-PL/SWCNTs in mice after intravenous injection. (a) Raman spectra of blood samples after the
injection of l-PEG2000-PL/SWCNTs (l: linear); (b) Raman spectra of blood samples after the injection of l-PEG5000-PL/SWCNTs; (c) Raman
spectra of blood samples after the injection of br-PEG7000-PL/SWCNTs; (d) PEG-PL/SWCNTs concentrations in blood at different time
points postexposure. Adapted from reference [31] with permission.

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The studies of other nanoparticles suggest that
covalent PEGylation is more efficient than noncova-
lent PEGylation [30]. This is true for CNTs, too. We
performed a pharmacokinetics
PEG-SWCNTs (covalently PEGylated SWCNTs) in
mice [35]. After PEG1500 was covalently linked to the
carboxyl groups of SWCNTs, the blood circulation
half-life of SWCNTs increased to 15.3 hours. Even
short PEG chains can prolong the circulation half-life
of CNTs efficiently while using covalent PEGylation.
Correspondingly, the
PEG1500-SWCNTs was reduced. The hepatic accumu-
lation level was 19.1 %ID/g for PEG1500-SWCNTs. The
good pharmacokinetics profile of PEG-SWCNTs
makes them promising in biomedical applications.
However, the biomedical
PEG-SWCNTs are fewer
PEG-PL/SWCNTs.





evaluation of
RES capture of
applications
than
of
of those

Figure 3. Time-dependent PEG-SWCNT concentrations in blood
(a) and mice (b) after the intravenous injection to mice. Adapted
from reference [35] with permission.

There might be two reasons: Firstly, the synthe-
sis of PEG-SWCNTs is much more difficult than that
of PEG-PL/SWCNTs, which limits the involvement of
more researchers. Secondly, covalent PEGylation de-
stroys the intact nature of SWCNTs, making some
unique properties of SWCNTs lost. After covalent
PEGylation, SWCNTs have strong fluorescence,
which hides the Raman signal of SWCNTs [36]. The
covalent functionalization also destroys the NIR flu-
orescence signal of SWCNTs. Still, drug delivery sys-
tems based on PEG-CNTs have been developed at
cellular level [37, 38] and in vivo level [39].
Another kind of functionalized CNTs was de-
veloped by Prato and coworkers [40]. CNTs were
functionalized via cycloaddition reactions, where
functional groups were directly attached to the carbon
skeleton of CNTs. Singh et al. reported the pharmaco-
kinetics and biodistribution of amino-CNTs [41].
Amino-CNTs were removed from body very quickly.
A small amount of injected amino-CNTs were
trapped in kidneys (20%ID/g), skin (9%ID/g), muscle
(8.5%ID/g) and lungs (1.3%ID/g). At 3 hours post
injection, only 2%ID of amino-CNTs were detected in
body. Another evaluation was performed by
McDevitt et al. [42, 43]. They reported that ami-
no-SWCNTs remained in body for more than 24
hours. At 3 hours postexposure, amino-SWCNTs ac-
cumulated in liver (17.8%ID/g), spleen (14.3%ID/g),
kidneys (8.3%ID/g) and skin (2.3%ID/g). Except in
kidneys, amino-SWCNT contents kept unchanged
after 24 hours. The different CNT sources and syn-
thesis conditions might attribute to the contradictory
results of the two groups. Such differences would lead
to different CNT lengths, functionalization degree
and so on. Due to the fast clearance of amino-CNTs
from blood, the in vivo applications of amino-CNTs
still require more efforts to improve their pharmaco-
kinetics profile, though various demonstrations have
been established at cellular level [44, 45]. However,
the amino-CNTs showed potential in delivering
siRNA to tumor via intratumoral injection [46] and to
brain via injection in the primary motor cortex [47].
For future studies, prolonging the side chains of
amino functional groups might be helpful to improve
the pharmacokinetics.
Other CNT samples tested show less potential in
biomedical areas. We have studied the pharmacoki-
netics of hydroxylated SWCNTs (SWCNTols) [23, 48]
and taurine functionalized MWCNTs (tau-MWCNTs)
[18]. SWCNTols distributed rapidly throughout the
whole body at 2 min postexposure to mice via tail
intravenous injection. The blood circulation half-life
of SWCNTols was around 10 min [48]. The migration
of SWCNTols in body seemed nearly free, that was

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also evidenced by very similar distribution profiles
after the exposures by different injection methods
[23]. The very short circulation time and free migra-
tion will limit the bio-applications of SWCNTols. On
the other hand, tau-MWCNTs targeted liver quickly
and stayed long there for months. The hepatic uptake
of tau-MWCNTs was higher than 80%ID (% of in-
jected dose) [18, 26], while the circulation half-life was
around 12 min [26]. The short circulation time and
high RES uptake indicate the difficulty of the bioap-
plications of tau-MWCNTs. Tyrosine functionalized
MWCNTs suffer the same problem as tau-MWCNTs
do [49].



Figure 4. Biodistribution of two different amino-SWCNTs in
mice after intravenous injection. Insets show the schematic
structures of amino-SWCNTs. Adapted from reference [41] with
permission.


As discussed above, the pharmacokinetics of
CNTs is very important for the biomedical applica-
tions. There are many parameters regulating the
pharmacokinetics of CNTs, such as size, charge and
surface chemistry. For CNTs, when the length is
smaller than 2 μm, CNTs can escape the pulmonary
capillary vessels and their pharmacokinetics is mainly
determined by the surface chemistry. Among the re-
ported surface chemistry, PEGylation is a most effi-
cient method in improving the pharmacokinetics pro-
file of CNTs. The development of easy, cheap and
reproducible PEGylation techniques becomes a cru-
cial route for the biomedical applications of CNTs.
Metabolism and transformation of CNTs
CNTs employed for biomedical purposes are in
forms of either dispersed or functionalized ones. After
introducing to biosystems, it is possible that these
CNTs are metabolized into other substances. The
metabolism of CNTs definitely affects their biomedi-
cal applications and also might lead to unwanted
toxicity. Therefore, the in vivo metabolism of CNTs
should be highly concerned. Unfortunately, the me-
tabolism of carbon nanomaterials is very hard to
study, because of the lack of proper analytical tech-
niques. Only few pilot studies are available in litera-
ture.
The skeleton of CNTs is relatively more stable
than their functional groups. We have shown the
transmission electron microscopy (TEM) images of
SWCNTs and MWCNTs (both pristine and function-
alized) trapped in vivo for more than 4 weeks. The
trapped CNTs even stayed as long as 3 months [18,
20]. The typical tube like or linear structure of CNTs
was clearly recognized under TEM, suggesting that
the carbon skeletons of CNTs were very stable against
in vivo metabolism as well as the subsequent acid
treatment for preparing the TEM samples. During the
purification and shortening processes, CNTs would
go through strong acid (HNO3, HCl and H2SO4)
treatment, sometimes assisted by ultrasonication.
Under such harsh conditions, the carbon skeleton
only breaks at the injured defects. The stability of
CNTs is also reflected by the long-term accumulation
in vivo without being metabolized, where CNTs were
visualized in mouse organs upon TEM at several
months postexposure [18, 20, 31]. Unlike most com-
mon results, Allen et al. observed the biodegradation
of SWCNTs through enzymatic catalysis in vitro [50].
SWCNTs were incubated in PBS (pH 7.0) in presence
of horseradish peroxidase and H2O2 in dark. After 16
weeks degradation, the loss of tube structure was
identified by TEM, thermogravimetric analysis
(TGA), matrix-assisted laser desorption-ionization
time-of-flight mass spectrometry (MOLDI-TOF MS),
dynamic light scattering (DLS), nanotube density of
states band diagram and UV-vis-NIR spectra. For the