Advanced Drug Delivery Reviews 62 (2010) 633–649
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r
DNA and carbon nanotubes as medicine☆
William Cheung, Francesco Pontoriero, Oleh Taratula, Alex M. Chen, Huixin He ⁎
Chemistry Department, Rutgers University, Newark, NJ 07102
a r t i c l e i n f o a b s t r a c t
Article history: The identiﬁcation of disease-related genes and their complete nucleotide sequence through the human
Received 10 November 2009 genome project provides us with a remarkable opportunity to combat a large number of diseases with
Accepted 3 February 2010 designed genes as medicine. However, gene therapy relies on the efﬁcient and nontoxic transport of
Available online 23 March 2010
therapeutic genetic medicine through the cell membranes, and this process is very inefﬁcient. Carbon
nanotubes, due to their large surface areas, unique surface properties, and needle-like shape, can deliver a
large amount of therapeutic agents, including DNA and siRNAs, to the target disease sites. In addition, due to
Nonviral delivery their unparalleled optical and electrical properties, carbon nanotubes can deliver DNA/siRNA not only into
Multifunctional cells, which include difﬁcult transfecting primary-immune cells and bacteria, they can also lead to controlled
Near infrared ﬂuorescence (NIR) release of DNA/siRNA for targeted gene therapy. Furthermore, due to their wire shaped structure with a
Raman diameter matching with that of DNA/siRNA and their remarkable ﬂexibility, carbon nanotubes can impact on
Carbon nanotubes the conformational structure and the transient conformational change of DNA/RNA, which can further
Single walled carbon nanotubes (SWNTs) enhance the therapeutic effects of DNA/siRNA. Synergistic combination of the multiple capabilities of carbon
Multiwalled carbon nanotubes (MWNTs)
nanotubes to deliver DNA/siRNAs will lead to the development of powerful multifunctional nanomedicine to
treat cancer or other difﬁcult diseases. In this review, we summarized the current studies in using CNT as
Small interference RNA (siRNA)
unique vehicles in the ﬁeld of gene therapy.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
1.1. DNA and Gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
1.2. Carbon Nanotubes: Structure, Physical and Chemical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
2. Delivery of therapeutic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
2.1. Nanocarrier approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
2.2. Chemical Modiﬁcation Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
3. Carbon Nanotubes (CNTs) as Novel Multifunctional Nonviral Gene Delivery Vehicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
3.1. Remarkable Capability in Delivery of DNA/siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
3.2. Unique Cell Internalization Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639
3.3. CNTs Impact on DNA Conformation and Second Conformational Transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641
3.4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
4. Challenges and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
4.1. In vivo targeted delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
4.2. In vivo permeability and circulation behavior of CNT/DNA/siRNA complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
4.3. Impact of CNTs on the conformation and conformation transition of DNA/siRNA for enhanced therapeutic effects . . . . . . . . . . . 646
4.4. Toxicity studies of carbon nanotubes with well-deﬁned and well characterized structures . . . . . . . . . . . . . . . . . . . . . . 646
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647
☆ This review is part of the Advanced Drug Delivery Reviews theme issue on "From Biology to Materials: Engineering DNA and RNA for Drug Delivery and Nanomedicine".
⁎ Corresponding author.
E-mail address: email@example.com (H. He).
0169-409X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
634 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
1. Introduction several hundred nanometers to several micrometers and diameter of
0.4-2 nm for single-walled carbon nanotubes (SWNTs) and 2-100 nm
1.1. DNA and Gene therapy for multi-walled carbon nanotubes (MWNTs). Conceptually, the
structure of SWNTs can be viewed as “wrapping-up” a graphene sheet
Sequencing of the human genome and functional genomics offer into a seamless hollow cylinder (Graphene is a one-atom-thick planar
unprecedented opportunities to combat a large number of diseases sheet of sp2-bonded carbon atoms that are densely packed in a
with designed genes either in the form of therapeutic oligonucleotides honeycomb crystal lattice.). The structure of MWNTs can be pictured
(ONs) or plasmids DNA carrying gene sequences . Gene expression as several co-axially arranged SWNTs of different radii with an inter-
can be disrupted at the transcriptional (triplex DNA) [2,3] or tube separation close to the inter-plane separation in graphite (0.34 -
translational (antisense DNA or short inference RNA) level [4–9]. In 0.35 nm) (Fig. 1) . There are an inﬁnite number of ways of rolling a
the triplex DNA-based antigene approach, transcription is disrupted graphene sheet into a cylinder. The large variety of possible helical
by the binding of a triplex-forming oligonucleotide at the promoter geometries, deﬁning the tube chirality, provides a family of nanotubes
region of a target gene . In the antisense strategy, the ON molecule with different diameters and chirality, which determined the most
corresponding to a target gene is delivered inside a cell where it binds signiﬁcant physical properties of SWNTs . The tubes are usually
complementarily with targeted messenger RNA (mRNA), producing a labeled in terms of the graphene lattice vectors by a pair of indices (n, m)
partially double-stranded ON/mRNA complex. Translation of this called the chiral vector (Fig. 2). The integers n and m denote the number
modiﬁed mRNA into protein is blocked by cleavage through the at- of unit vectors along two directions in the honeycomb crystal lattice of
traction of Ribonuclease H towards hybridized ON/mRNA complex or graphene . For a given (n,m) nanotube, if n = m, or if n − m is a
by steric hindrance of the antisense molecule which prevents mRNA multiple of 3, the nanotube is metallic, otherwise the nanotube is a
recognition by ribosomes [10,11]. Due to extensive work in this ﬁeld, semiconductor [24,25]. It is worth to mention that MWNTs are
there is already one antisense ON product approved for local therapy of essentially metallic in nature due to the inter-tube interactions .
cytomegalovirus retinitis (Vitravene) and nearly twenty others in late- Carbon nanotubes have excellent electronic properties: metallic
stage clinical trials . Despite the successes mentioned above, nanotubes can carry an electrical current density of 4 × 109 A/cm2,
folding of target RNAs or their association with speciﬁc proteins in the which is three orders of magnitude higher than a typical metal, such as
cell often prevent the ON molecules from binding to their targets. This copper or aluminum . Individual semiconducting SWNTs are known
requires employment of relatively high doses in order to achieve a to possess an extremely high carrier mobility of 10 000 cm2 /Vs at room
therapeutic effect. These high doses, as well as the consequently high temperature, and can be operated at high frequencies (2.6 GHz). These
toxicity that is sometimes observed, have made ON molecules less values exceed those for all known semiconductors, such as silicon ,
attractive for therapeutic product development [13,14]. which bodes well for application of nanotubes in high-speed transistors,
The rapid development of mammalian RNA interference (RNAi) single- and few-electron memories, and chemical/ biochemical sensors
opens the path to a powerful new strategy for therapeutic regulation [29,30]. Moreover, they are ﬂexible owing to their small diameter.
of gene expression [7,15,16]. It is an evolutionarily conserved process SWNTs are therefore also an ideal candidate material for high-
by which double-stranded small interfering RNA (siRNA) induces performance, high-power, ﬂexible electronics [31,32]. Carbon nano-
sequence-speciﬁc, post-transcriptional gene silencing . The tubes are also the strongest and stiffest materials yet discovered in terms
revolutionary ﬁnding of RNAi resulted from the work of Andrew of tensile strength and elastic modulus respectively. The Young's
Fire and coworkers, who demonstrated in 1998 that injection of long modulus is over 1 Tera Pascal. It is stiff as diamond. The estimated
double stranded RNA (dsRNA) into the nematode C. elegans sequence tensile strength is 200 Giga Pascal . The strength results from the
speciﬁcally induced silencing of homologous genes . The key to covalent sp² bonds formed between the individual carbon atoms and
potential applications of RNAi therapy in humans was the discovery these properties are ideal for reinforced composites [34–37], nanoelec-
that employment of short dsRNA (b 30 bp) resulted in bypassing the tromechanical systems (NEMS) . Furthermore, the heat transmis-
mammalian immune response and facilitating gene-speciﬁc silencing sion capacity of individual CNTs at room temperature has been shown to
. This discovery leads to the realization that synthetic siRNA exceed 3000Wm-1 K-1, which is greater than natural diamond, excellent
designed with a sequence complementary to a target gene could be for thermal management . Equally important, both SWNTs and
delivered to cells instead of long dsRNA. A simpliﬁed model for the MWNTs are now produced in substantial quantities for these varieties of
RNAi mechanism or pathway is based on two steps. In the ﬁrst step, commercial applications.
short double stranded RNA is introduced to the cytoplasm. In the
second step, siRNAs are loaded into the effector complex RNA-
induced silencing complex (RISC). The siRNA is unwound during RISC
assembly and the single-stranded RNA hybridizes with the mRNA
target. Gene silencing is a result of nucleolytic degradation of the
targeted mRNA by the RNase H enzyme Argonaute 2.
There is increasing enthusiasm for developing therapies based on
RNAi [5–7,9]. The advantage of RNAi compared to other gene
therapeutic strategies lies in its high afﬁnity and speciﬁcity to their
target sites and its high potency to silence the targeted genes [6,9].
Promising results have been attained with siRNAs in animal models
[5,17,18] and several clinical trials  are underway. However, just
like other gene therapy strategies, the main obstacle to the success of
siRNA therapeutics is in delivering siRNAs across the cell membrane
to the cytoplasm where it can enter the RNAi pathway and guide the
sequence-speciﬁc mRNA degradation [5,9,19–22].
1.2. Carbon Nanotubes: Structure, Physical and Chemical Properties
Fig. 1. Conceptual diagram of (A) single-walled carbon nanotube and (B) multi-walled
Carbon nanotubes (CNTs) are well-ordered, all carbon hollow carbon nanotube showing typical dimensions of length, width, and inter-tube
graphitic nanomaterials with very high aspect ratios, lengths from separation in multi-walled carbon nanotubes. The ﬁgure was adapted from Ref. .
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 635
interested in this area should reference these works. In this review, we
will focus on CNTs as unique gene delivery vehicles and mediators for
gene therapy. We start with a short summary of the current gene
delivery approaches, followed by the unique capabilities exhibited by
CNTs in delivery of DNA and siRNA. Lastly, we summarize the major
advantages, opportunities, and challenges ahead for clinical applica-
tion of CNT in gene therapy.
2. Delivery of therapeutic genes
Studies in the realm of non-viral gene therapy based on plasmid
DNA (pDNA) and antisense ONs have been ongoing for years and will
continue toward improving systemic delivery and transfection
efﬁciencies to the levels required for in vivo clinical trials. DNA and
siRNA are double-stranded nucleic acids and both contain anionic
phosphodiester backbones with the same negative charge to
Fig. 2. Graphene honeycomb network with lattice vectors a1 and a2. The chiral vector
Ch = ma1+ na2 represents the possible wrapping of the two-dimensional graphene
nucleotide ratio and can interact electrostatically with cationic agents.
sheet into a tubular form. The direction perpendicular to Ch is the tube axis. In the Therefore, delivery of siRNA can borrow the knowledge learned from
present example, a (5,3) ( m = 5, n = 3) nanotube is under construction and the the longer-studied problems of DNA in vitro and in vivo delivery
resulting tube is illustrated on the right. The ﬁgure was reproduced from Ref.  with [1,54–56]. Despite superﬁcial similarities, one must keep in mind that
siRNA molecules possess distinct characteristics, and delivery tech-
nologies should be developed to suit this case speciﬁcally .
In recent years, efforts have also been devoted to exploring the Recently, Juliano et al. surveyed and summarized the mechanism and
potential biological applications of CNTs, motivated by their interest- strategies for effective delivery of antisense and siRNA oligonucleo-
ing size, shape, and structure, as well as attractive optical and tides . There are several obstacles which have to be overcome in
electrical properties [40,41]. First, with all atoms exposed on the order to achieve the sufﬁcient delivery of siRNA molecules into the
surface, SWNTs have ultrahigh surface area (theoretically 1300 m2/g) targeted cancer cells. First is the degradation by serum and tissue
that permits efﬁcient loading of multiple molecules along the length nucleases. Unlike DNA, the RNA backbone contains ribose which has a
of the nanotube sidewall. Second, supramolecular binding of aromatic hydroxyl group in the 2′ position of the pentose ring instead of a
molecules can be easily achieved by π-π stacking of those molecules hydrogen atom . It makes the RNA backbone more susceptible to
onto the polyaromatic surface of nanotubes . Moreover, the hydrolysis by serum nucleases in extracellular environment which
ﬂexible 1-D nanotube may bend to facilitate multiple binding sites of a cleave along the phosphodiester backbone of nucleic acids. It is
functionalized nanotube to one cell, leading to a multi-valence effect, important to notice that siRNA fragments of less than 21 base pairs
and improved binding afﬁnity of nanotubes conjugated with targeting have been shown to have a less potent RNAi effect . The second
ligands . hurdle is the rapid excretion via kidney due to the size of siRNAs:
The intrinsic optical and electrical properties of SWNTs can be these molecules are relatively small and thus are rapidly excreted
utilized for multimodality imaging and therapy. Due to quantum through urine when administrated into the blood stream, even
conﬁned effects, SWNTs behave as quasi 1-D quantum wires with though the siRNA molecules remain stable . The third challenge is
sharp densities of electronic states (electronic DOS) at the van Hove the inefﬁcient endocytosis by targeted tumor cell, and the inefﬁcient
singularities, which impart distinctive optical properties to SWNTs release from endosomes. Viruses have evolved functions to efﬁciently
. SWNTs are highly absorbing materials with strong optical overcome these barriers, however, the immune response elicited by
absorption in the near-infrared (NIR) range (800-1600 nm). These viral proteins has posed a major challenge to this approach . There
wavelengths include the tissue transparent region of the electromag- is much interest in developing nonviral gene delivery vehicles 
netic spectrum (800-1400 nm), in which radiation passes through which transfer siRNA therapeutics speciﬁcally to the treatment area
without signiﬁcant scattering, absorption, heating, or damage to and can bypass the cell membrane to release payload inside the cell
tissues. Therefore, SWNTs can be utilized for photothermal therapy [55,60,61]. Currently there are several types of nonviral delivery
[45–47] , and photoacoustic imaging . SWNTs can also be used to systems under investigation which could improve the serum stability
deliver therapeutic drugs with externally controlled release capabil- and cellular internalization of siRNA molecules. The known nonviral
ities . delivery systems could be classiﬁed into two groups: nanocarrier
Furthermore, semiconducting SWNTs with small band gaps on the approach and chemical modiﬁcation approach.
order of 1 eV exhibit photoluminescence in the NIR range. The
emission range of SWNTs is 800∼2000 nm [41,49,50], which covers 2.1. Nanocarrier approaches
the biological tissue transparency window, and is therefore suitable
for biological imaging. SWNTs also have distinctive resonance- Cationic lipids and polymers are two major classes of nonviral
enhanced Raman signatures for Raman detection/imaging, with DNA/siRNA delivery carriers that are positively charged and can form
large scattering cross-sections for single tubes [51,52]. The selective complexes with negatively charged DNA/siRNA [60,62,63]. The
detection of diseased cells and tissues by the use of nanotubes that can nucleic acids can be compacted into a tiny nanoparticle with size
insert themselves into such areas of interest may possibly provide for ∼50-200 nm [1,64–67], allowing complete protection of the nucleic
a more sensitive and localized diagnostic approach . In summary, acid from nuclease degradation. It can carry a large “payload”
motivated by various properties of CNTs, research towards applying comprising multiple copies of DNA/siRNA. They can be modiﬁed
carbon nanotubes for biomedical applications has been progressing with multiple copies of targeting ligands, thus providing high afﬁnity
rapidly. Very recently, Liu and Dai et al.  gave a comprehensive with the target cells. Nanoparticles can be designed to release their
review on this ﬁeld and clariﬁed that surface functionalization is contents at prescribed rates and can also be engineered to assist in the
critical to the behavior of carbon nanotubes in biological systems. release of their contents from endosomes. Some of these formulations
Kostarelos et al.  also summarized the promises, facts and current have demonstrated high transfection efﬁciency in vitro . However,
challenges for carbon nanotubes in imaging and therapeutics. Readers for systematic in vivo application, the nanocarrier approaches faces
636 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
other hurdles: First, despite advances in using PEG or other various nanomaterials, carbon nanotubes are highly promising for
hydrophilic polymers for extracellular stability to prolong their delivery of drugs, including therapeutic nucleic acids due to their
circulation in the blood stream, a large fraction of the injected dose unique size, shape, and physicochemical properties as discussed in the
of nanoparticles will accumulate in the liver and be taken up by previous section. These materials can now be functionalized to a
hepatic phagocytes. Second, due to the vascular endothelial barrier, sufﬁcient degree to facilitate nearly complete excretion of SWNTs
nanoparticles can only reach certain tissues such as the liver, spleen, from mice over time . In the following section, the application of
and some types of tumors due to enhanced permeability and CNT in the delivery of nucleic acids, including DNA, RNA, and siRNA
retention (EPR) effect , where the nanoparticles tend to will be summarized. Furthermore, the unique capability of CNTs in
accumulate in tumor tissues much more than in normal tissues. inﬂuencing the conformation and conformational transient change of
However, nanoparticles cannot or very difﬁcult to access parenchymal DNA and RNA, therefore their therapeutic effects will be discussed.
cells in most normal tissues; they are simply excluded by the
endothelial barrier. Thus many potential disease targets cannot be 3. Carbon Nanotubes (CNTs) as Novel Multifunctional Nonviral
addressed by the nanocarrier approaches. Gene Delivery Vehicles
2.2. Chemical Modiﬁcation Approaches 3.1. Remarkable Capability in Delivery of DNA/siRNA
For siRNA, triplex antigen, or antisense ON therapeutic ap- The ﬁrst work of utilization of carbon nanotubes as a novel
proaches, the stability of these nucleic acids in the extracellular and gene delivery vector system was reported by Bianco et al. .
intracellular environments can also be improved by a variety of They covalently modiﬁed carbon nanotubes using the Prato reaction,
chemical modiﬁcation methods, including changes in the oligonucle- a method based on the 1,3-dipolar cycloaddition of azomethine ylides
otide backbone, replacement of individual nucleotides with nucleo- (Sch.1). Both single walled carbon nanotubes (SWNTs) and multi-
tide analogues and addition of conjugates to the oligonucleotides walled carbon nanotubes (MWNTs) were functionalized with a
[60,62]. Such chemical modiﬁcations must be designed such that they pyrrolidine ring bearing a free amine-terminal oligoethylene glycol
do not interfere with the recognition ability to their target gene and moiety attached to the nitrogen atom. The presence of this functional
should also not interfere with subsequent reactions, for example the group increases the solubility of carbon nanotubes in aqueous
degradation of the target mRNA. One of the simplest and most solutions. Small bundles of nanotubes were formed with a diameter
promising modiﬁcations is the introduction of phosphoro-thioate of around 20 nm and length of around 200 nm. The delivery of
(PS) linkages, which are known to reduce siRNA cleavage by nuclease. plasmid DNA (pDNA) and the expression of β-galactosidase (marker
However, siRNAs with extensive PS linkages are also known to gene) in CHO cells were studied. Like other nonviral gene delivery
increase binding to serum proteins and can be toxic in vivo . vectors, the amine functionalized nanotube was able to condense
Another apparently useful modiﬁcation is preparation of 2’-O-methyl, plasmid DNA to form supramolecular complexes with globular
2’-ﬂuoro, 2’-O-(2-methoxyethyl) and lock nucleic acid nucleotides conformations through electrostatic interactions . It was also
[70–75]. Additionally, bioconjugation of one or both strands of siRNAs found that the charge ratio between the ammonium groups at the
with lipids, polymers, and cell penetrating peptides is often desirable SWNT surface and the phosphate groups of the DNA backbone was an
to further increase their thermodynamic and nuclease stability, important factor determining the level of gene expression. The
improve the biodistribution and pharmacokinetic proﬁles of siRNAs, expression was only 10 times higher than the naked pDNA alone,
and target them to speciﬁc cell types [60,62]. The best advantage of still much less effective than that of liposomes.
the chemical modiﬁcation approach is the relatively small sizes of the However, they found that the DNA carbon nanotube (DNA-CNT)
products which causes a fundamental difference in their in vivo complexes does not exert any mitogenic or any toxic effect on the
behavior as compared to the nanocarrier approach. For example, the activated or nonactivated lymphocyte, which is very different from
size of the conjugates may still be far smaller than the pores in normal other nonviral gene deliver vectors such as dendrimers and liposomes.
vascular endothelium, thus in principle they should be able to access These traditional nonviral gene deliver vectors generally cause
virtually all tissues. However, it may encounter the problem of rapid destabilization of the cell membrane and lead to pronounced
excretion via the kidney when administrated into the blood stream, cytotoxicity while achieving effective delivery of DNA. They attributed
despite the fact that siRNA/antisensor ON molecules remain stable . the lower cytotoxicity of the DNA-CNT complex to the capability of
Furthermore, each conjugate requires a separate synthesis, whereas in penetrating cell membrane. They studied the internalization mecha-
the nanocarrier approaches, one nanoparticle can potentially accom- nism of the amine functionalized carbon nanotubes and found that
modate a variety of different oligonucleotides. Lastly, only a single probably these carbon nanotubes entered the cells by a spontaneous
ligand can be conjugated to the oligonucleotide, therefore the afﬁnity mechanism in which they behaved like nanoneedles and passed
interaction with the targeted receptors will be much lower than the through the cell membrane without causing cell death. The hypothesis
case for multivalent nanoparticles. Another key issue is the release
from endosomes upon cell uptake.
Thus, both nanocarriers and molecular conjugates exhibit advan-
tages and disadvantages as delivery strategies. Ultimately, the most
attractive delivery system may turn out to be neither a relatively small
monomolecular oligonucleotide conjugate nor a large nanoparticle.
Rather it may be an intermediate-sized moiety, perhaps comprised of
oligonucleotides and targeting agents covalently linked to a small
polymer  or protein that is large enough to avoid rapid excretion
but yet small enough to be able to pass the vascular endothelial
barrier. This approach may offer some of the high payload and high-
afﬁnity targeting aspects of nanoparticles without the constraints due
to relatively large particle size. Another attractive approach is to
explore the remarkable physical properties and the small size of Scheme 1. Azomethineylides functionalized carbon nanotube. The ﬁgure was
various nanomaterials to develop a new delivery concept to overcome reproduced from Ref. with permission. The ﬁgure was reproduced from Ref. 
all of the mentioned delivery barriers for efﬁcient therapy. Among with permission.
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 637
was supported by a recent publication with molecular dynamics (20 bp) was complexed with ammonium-functionalized SWNTs, and
simulation, which suggested that hydrophobic nanotubes with then the obtained complex was mixed with phospholipid-PEG
hydrophilic functional groups can spontaneously insert into a lipid containing a tumor targeting moiety (folic acid), followed by soni-
bilayer. They believed that a semi-rigid and elongated form of the cation in an ice bath for 30 min and agitation in dark for 24 hours. The
carbon nanotubes rules out an endocytosis process. This deduction experimental results show that the obtained multifunctional DNA-
was also conﬁrmed by an experiment which showed that the SWNT complex has good tumor cell targeting property. However, the
internalization was not affected by pre-incubation of the cells with therapeutic effects of this targeting DNA-SWNT complex have not
sodium azide or 2,4-dinitrophenol, typical inhibitors of energy- been tested yet.
dependent cell process such as endocytosis. However, similar study To increase the efﬁciency of DNA condensation, Liu group in China
was not performed to study the internalization mechanism of the  modiﬁed chitosan with β-cyclodextrin and pyrene derivatives.
DNA-CNT complexes. They believed that these complexes are able to This modiﬁed chitosan wrapped around multiwalled carbon nano-
bind to, and penetrate within the cells through an endosome- tubes (MWNTs). The DNA condensation capability of was validated by
independent spontaneous insertion mechanism, similar to the amine AFM and dynamic light scattering, and found that grafting of pyrene
functionalized carbon nanotube alone before DNA complexion. moieties along the chitosan chain pronouncedly improved the
Furthermore, they did not mention if the pDNA was released from efﬁciency. The cooperation between cationic and aromatic groups is
the carbon nanotube surface for the efﬁcient gene expression. In a the key factors to enhance DNA condensation.
separate study, they demonstrated that functionalized CNT can greatly Also with a goal for efﬁcient delivery of DNA by carbon nanotubes,
improve the immunostimulatory properties of CpG containing ONs in Liu group in Singapore  grafted multiple polyethylenimine onto
vitro, which was also attributed to the high loading capacity and cell the surface of MWNTs. They demonstrated that the transfection
penetrating ability of the amine functionalized CNT . efﬁciency was three times higher than that of PEI (25 K) and four
Along a similar line, Xu et al. applied ammonium-functionalized orders of magnitude higher than that of naked DNA. They labeled PEI-
SWNTs, which have been used to deliver siRNA targeted to cyclin A2 in MWNTs with ﬂuorescein isothiocyanate (FITC). Using confocal
chronic myelogenous leukemia K562 cells, resulting in suppression of microscope imaging, they demonstrated that the DNA complexed
cyclin A2 expression . The depletion of cyclin A2 causes cell with the ﬂuorescently labeled PEI-MWNTs entered cells after
proliferation arrest and promotes apoptosis of chronic myelogenous incubation for 1 h at 37 ° C, but only very weak green ﬂuorescence
leukemia K562 cells. The ammonium-functionalized SWNTs was also could be detected after incubation for 1 h at 4 ° C. Based on this
employed to mediate the delivery of telomerase reverse transcriptase temperature-dependent cell uptake, they concluded that the uptake
(TERT) siRNA into tumor cells , wherein they released the siRNAs of the DNA-PEI-MWNTs complexes was through endocytosis. The
to silence the targeted TERT gene, which is critical for the devel- high transfection efﬁciency of PEI-MWNTs was attributed to several
opment and growth of tumors. The treatment with the SWNT-TERT- factors. The ﬁrst factor is the secure immobilization of DNA onto the
siRNA complexes led to the suppression of the cancer cell growth. By surface of MWNTs which leads to the formation of stable complexes
injection of this complex in mice bearing Lewis lung carcinoma tumor that protected DNA from degradation. The second factor is that the
or HeLa cell xenografts, tumor growth was inhibited and the average proton-sponge effect of the grafted PEI would allow the DNA-PEI-
tumor weight was signiﬁcantly reduced when compared to that of the MWNTs complexes to escape easily from endosomes or other vesicles
untreated animals. This work is one of the three reports currently in in cells, which have been well documented . Furthermore, the
the literatures using SWNT mediated nucleic acid in vivo delivery and larger complexes of DNA-PEI-MWNTs would improve the proton-
treatment. Very Recently, Kostarelos et al. reported another in vivo sponge effects of PEI and facilitate a more effective sedimentation
siRNA treatment of a human lung carcinoma model delivered by onto the cells . In 293 cells, the complexes of DNA-PEI-MWNTs
ammonium-functionalized MWNTs. The results demonstrated that with a weight ratio of 10:1 showed no signiﬁcant effects on cellular
MWNT-NH3:siRNA complexes were active by triggering an apoptotic metabolism but higher ratios led to a decreased cell number. Pure PEI-
cascade, leading to extensive necrosis of the human tumor mass and MWNTs showed a higher cytotoxicity. The cytotoxicity of PEI is
increased survival of tumor-bearing animals. This work provided the related to the molecular weight: a higher molecular weight results in
ﬁrst comparative in vivo study against a ‘benchmark’ nanoparticles a higher cytotoxicity . PEI-MWNTs may behave as high molecular
with a proven clinical record, such as cationic liposomes. They also weight PEI and thus should have a certain degree of cytotoxicity.
found that the MWNT-NH3:siRNA complexes were more effective in Similar principle was also applied to intracellular delivery of quantum
prolonging the survival of tumor-bearing animals, presumably owing dots tagged antisense ON by PEI modiﬁed MWNTs and siRNA by
to their more facile translocation into the tumor cell cytoplasm. Even hexamethylenediamine and poly(diallyldimethylammonium) chlo-
though the complexes were administrated by intra-tumoral local ride (PDDA) functionalized SWNTs respectively [89,90].
injection, the work is inspiring and encouraging further studies to Dai and colleagues have developed “smart” DNA/siRNA delivery
explore the unique capabilities of chemically functionalized carbon systems based on SWNTs [47,91,92]. In contrast to the approaches
nanotubes in gene delivery for the development of advanced thera- described above, DNA or siRNA cargos can be controllably released from
peutic formulation to ﬁght various diseases. the carbon nanotube surface upon cellular uptake for efﬁcient gene
Another SWNT mediated in vivo delivery of siRNA for tumor silencing. The ﬁrst of his work along this line is conjugation of antisense
immunotherapy was reported by Yang et al. . The SWNT func- ONs or siRNAs onto functionalized carbon nanotubes by incorporation
tionalization and siRNA complexation was similar to that approached of biologically triggered cleavable bonds . The ﬁrst step in this
by Wang et al. . However, they believed that phagocytosis was the approach involves making a stable aqueous suspension of short SWNTs
mechanism by which functionalized SWNTs enter into cells. Based on by noncovalent adsorption of phospholipids molecules with poly
the mechanism, they hypothesized that intravenously (i.v.) delivered (ethylene glycol) ( PL-PEG) chains and terminal amine or maleimide
SWNTs might be preferentially engulfed in vivo by antigene-present- groups. The PL-PEG binds strongly to SWNTs via Van der Waals and
ing cells, which possess phagocytic potential such as dendritic cells hydrophobic interactions between two PL alkyl chains and the SWNT
and macrophages. The experimental results indeed demonstrated sidewall, with the PEG chain extending into the aqueous phase to impart
that intravenous injection of siRNA-SWNT complexes signiﬁcantly solubility in water. The suspension is extremely stable in PBS buffer even
retarded tumor growth after 15 days while siRNA alone or mock upon heating to 70 ° C for weeks. Thiol-modiﬁed DNA or siRNA cargo
siRNA-SWNTs complex has no signiﬁcant effect. Taking another step molecules were linked to the amine or maleimide groups on the
further, this group developed an approach for targeted delivery of sidewalls of SWNTs through cleavable disulﬁde bonds, which can be
DNA mediated by SWNTs  (Scheme 2). First, double stranded DNA cleaved by thiol reducing enzymes thus releasing the cargos from the
638 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
Scheme 2. Preparation of functionalized SWNTs for targeted delivery of DNA into tumor cells. The ﬁgure was reproduced from Ref.  with permission.
SWNT once the conjugates are internalized into the endosomal or cells can trigger endosomal rupture which releases the noncovalent
lysosomal compartments through endocytosis (Fig. 3). The released wrapped DNA from the nanotube surface .
cargos can freely reach their intended biological destinations. Electroporation is a technique in which bio-membrane are per-
However, without disulﬁde linkage (Fig. 3A, 2-X) the DNA cannot meabilized by pulsed electric ﬁelds of several kV cm-1 amplitude and
be released to the cell nucleus. They also demonstrated higher submicrosecond duration. Thereby membrane pores occur temporar-
silencing efﬁciency when siRNAs were conjugated with SWNTs with ily and macro biomolecules, such as DNA or drugs, can be transferred
disulﬁde bonds, which were attributed to the active releasing of siRNA into living cells. However, existing electroporation technology is
from SWNTs by enzymatic disulﬁde cleavage, maximizing the limited in its ability to treat large quantities of cell materials and DNA.
endosome/lysosome escape of siRNA. The functionality of siRNA Additionally, the application of high electric ﬁeld pulses can lead to
may be less perturbed when in a free and released form than when irreversible electroporation and, consequently, cell lysis. Giersig et al.
attached to SWNT sidewalls. Recently they explored this approach to [95,96] reported that CNTs can be used as nanoscale “electroporation
deliver siRNA into human T cells and primary cells, which are difﬁcult vectors” to deliver plasmid DNA into bacteria, taking advantage of the
to transfect by traditional nanoviral agents such as liposomes . unique shape (large aspect ratio) and electronic properties of carbon
They found that the delivery ability and RNAi efﬁciency of these nanotubes. Under microwave irradiation, MWNTs create temporary
carbon nanotubes far exceed those of several existing nonviral transmembrane “nanochannels” that facilitate plasmid DNA delivery
transfection agents including four formulations of liposomes. The into cells. When placed in an electric ﬁeld, charges are induced on the
high delivery ability was attributed to the large surface area of SWNTs tip of the CNTs and the electric ﬁeld at the tips drastically enhanced by
for efﬁcient siRNA cargo loading, high intracellular transporting a factor of 10-100 depending on their length to diameter aspect ratios.
ability of SWNTs, and high degree of endosome/lysosome escape The charges on the tip and the strong electric ﬁeld induce charges on
owing to the intracellular cleavable disulﬁde conjugation approach. the cell surface, which leads to a tip-ﬁrst CNT attraction to the cell
In another work, they explored the unique physical and chemical wall. Consequently, localized sites of the cell envelope targeted by the
properties of SWNTs to control the release of DNA from the carbon electric ﬁeld created at the tip of individual CNTs are brought to a
nanotube surface upon internalization by endocytosis. In this work, permeabilized state. The electropermeabilization of the targeted sites
the DNA was physically wrapped around the SWNTs and was occurs during the microwave pulse, followed by the delivery of the
prepared according to simple procedures by Zheng et al. [93,94] plasmid to the cytosol. When the microwave irradiation is exhausted
(Fig. 4). Atomic Force Microscopy (AFM) was used to characterize the current stops and the magnetic ﬁeld collapses, then the
these DNA-SWNTs complexes. They were short (∼ 50-200 nm), permeabilized sites formed at the cell surface reseal spontaneously,
individual, and small bundles of nanotubes. They showed very strong and the cells can continue to grow. So the CNTs acted as transient
near IR absorptions and the absorptions increase as a function of dipoles allowing nanoscale cell targeting and gentle electroporation.
concentration of the tubes. NIR excitation of the SWNTs inside the Heating produced in this microwave irradiation was negligible. The
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 639
Fig. 3. (A) Two schemes of SWNT functionalization by thiolated biological molecule X with (1-X) and without (2-X) disulﬁde bond respectively. Both DNA and RNA cargos contain a
thiol functional group and a six-carbon long spacer at the 5' end of the DNA or RNA. (B) UV-vis-NIR of a 1-DNA solution (peak at ∼550 nm due to Cy3 label on DNA) and
(C) ﬂuorescence spectra (for Cy3 label) of a 1-DNA and 2-DNA, respectively, before (black) and after dithiothreitol (DTT) treatment and ﬁltration (blue for 2-DNA and red for 1-DNA).
The ﬁgure was reproduced from  with permission.
exposure of E. coli cells to short microwave irradiation and low process determine the transduction efﬁciency and contribute to the
concentrations of CNTs had no detrimental effect on cell viability or difference in transduction efﬁciency between primary and trans-
morphology. This technique is fundamentally different from tradi- formed cells. This method of gene delivery will enhance genetic
tional electroporation techniques used today because electrodes are transduction of exogenous DNAs, particularly in primary cells. In a
not needed. It also considerably reduces the amount of plasmid recent report, they further simpliﬁed the nanospearing technique to a
required for efﬁcient transformation. one step procedure and demonstrated the biocompatibility of this
Along a similar line, Cai et al.  proposed a novel spearing technique with respect to primary B lymphocytes . The results
technique for cellular internalization of carbon nanotubes and indicated that the nanospearing technique did not result in cellular
plasmid DNA. The carbon nanotubes contained ferromagnetic nickel toxicity nor perturb cellular homeostasis (non-speciﬁc activation of
catalyst particles enclosed on their tips and responded to magnetic primary cells).
agitation. The spearing technique involves a two step procedure. First
the cells and carbon nanotubes are exposed to a magnetic ﬁeld. This
allows the carbon nanotubes to spear the cell membrane. Next the 3.2. Unique Cell Internalization Mechanisms
cells are transferred to fresh medium and a static ﬁeld is applied that
enhances the spearing procedure and pulls the carbon nanotubes into Dai et al.  carried out a systematic investigation of the cellular
the cell. This technique proves to be a powerful tool for efﬁcient internalization mechanism and pathway for the DNA-SWNT com-
molecular delivery of plasmid DNAs. The delivery was found to be plexes and proposed an energy and clathrin-dependent endocytosis
highly efﬁcient with high viability which was attributed to the unique pathway. Endocytosis is known as a general entry mechanism for
delivery mechanism: nanopenetration of the cell membrane. Re- various extracellular materials and is an energy dependent uptake,
markably, the nanotube spearing technique requires very low which is hindered when incubations are carried out at low
concentration (100 fM compared to the 1-5 μM in the work by temperatures (4○ C instead of 37○ C) or in ATP (adenosine
endocytosis pathway) of nanotubes for efﬁcient transduction efﬁ- triphosphate) depleted environments. The SWNTs that were used
ciency. The use of magnetic force resulted in 107 fold improvement in for this study were short (∼ 50-200 nm), individual, and small
the molecular shuttling efﬁciency. The transduction efﬁciency of this bundles of nanotubes as characterized by Atomic Force Microscopy
delivery approach is also much higher than magnetofection, which (AFM). They incubated the SWNT conjugates with cells at 4○ C and
can also facilitate vector delivery using a magnetic ﬁeld. Furthermore, with cells pretreated with NaN3. NaN3 treatment is used to disturb
with nanotube spearing the nucleus may also be penetrated, and ATP production in cells. Confocal microscopy study of the cells
therefore received plasmid DNA directly from the invasive nanotubes. incubated at different conditions indicates that the cellular uptake
This is very different from traditional nonviral delivery methods such mechanism involves endocytosis. Cell ﬂow cytometry measurements
as liposome and polycationic polymer vehicles. Before the plasmid further supports this conclusion. Recent reports by Heller et al.
DNAs reach the nucleus, their intracellular trafﬁcking has to proceed [51,100] and Becker et al.  also demonstrated the same inter-
via the endosomal or lysosomal pathway, in which a large number of nalization pathway by exploiting the extremely stable NIR ﬂuores-
plasmids are hydrolyzed. The fates of the plasmids in this trafﬁcking cence and Raman properties of SWNTs.
640 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
Fig. 4. Carbon nanotubes with high NIR absorbance dispersed in water. (A) Schematic of a DNA-SWNT complex, in which the DNA wrapped around the SWNTs. (B) UV-visible NIR
spectra of solutions of individual SWNTs functionalized noncovalently by 15-mer Cy3 labeled-DNA at various nanotube concentrations (top curve, SWNT concentration ≈ 25 mg/
liter in H2O; lower curves correspond to consecutive 3% reduction in SWNT concentration). The well deﬁned peaks in the UV-visible NIR spectra suggest lack of large aggregated
SWNTs in the solution by removing bundles by centrifugation. (C) Absorbance at 808 nm vs. SWNT concentration (optical path = 1 cm). Solid line is Beer's law ﬁt to obtain molar
extinction coefﬁcient of SWNT ε ≈ 7.9 × 106 M–1·cm–1. (Inset) A photo of a DNA-functionalized SWNT solution. (D) AFM image of DNA-functionalized individual SWNTs (height of
1–10 nm) deposited on a SiO2 substrate. (Scale bar: 200 nm.) The ﬁgure was reproduced from Ref.  with permission.
It is worthy to mention that traditional transfection agents such as breaks down into multiple small vesicles early in mitosis, allowing the
cationic liposomes, peptides, and polymers can bind to the surface of translocation of SNWT-poly (rU) hybrids. In telophase, the last mitotic
immortalized cancer cells, which constitutes with high surface stage, the nuclear envelope reforms, possibly incorporating the
negative charges, through electrostatic forces to initiate cellular SNWT-poly (rU) hybrids. Cell growth and MTT assay have shown no
uptake and molecular delivery. However, they found that liposomes toxicity in either MCF 7 breast cancer cells or d2C keratinocytic cells
are incapable of delivery into T cells, suggesting that the electrostatic for concentrations up to 1 mg/mL over a 3 day period.
driving forces for cellular binding and uptake may not be generic to all There are several subcategories of the endocytosis pathway, such as
cell types . The delivery ability of nanotubes into T cells was phagocytosis, pinocytosis, clathrin-dependent receptor-mediated, and
discovered to be highly dependent on the degree of hydrophilicity of clathrin-independent endocytosis. To test if the mechanism is clathrin
the functionalized SWNTs, which causes different hydrophobic dependent, the cells were pretreated with sucrose or K+ depleted
interactions with the cells. Therefore, they proposed that hydrophobic medium prior to incubation with the SWNT conjugates. These pre-
interactions between nanomaterials and cell surfaces could be treatments are known to disrupt the formation of clathrin-coated
exploited as a more generic driving force for cellular binding and vesicles on the cell membrane. Cell cytometry results demonstrated
internalization. This mechanism is consistent with the report by Lu et that there was a drastic reduction in cellular uptake of SWNT conjugates
al. , in which cellular uptake of CNT-RNA (poly(rU)) complex (Fig. 5A). Transferrin, known to enter cells by the clathrin-mediated
formed through non-speciﬁc binding with the CNT was studied. The endocytosis pathway was also blocked from entering the cells when
uptake of the SNWT-poly (rU) is also hypothesized to be a result of incubated at low temperature and in K+ depleted buffer (Fig. 5B). On the
amphipathic properties of both the cellular membrane and the SWNT- other hand, the cells were pretreated with ﬁlipin and nystatin drugs to
poly(rU) hybrids. Lateral diffusion of the phospholipids within the disturb the cholesterol distribution within the cell membrane, which
biomembrane may contribute to the translocation of SNWT-poly (rU) can inhibit clathrin-independent endocytosis such as the caveolae or
hybrids by allowing hydrophobic interactions between SWNTs and lipid-rafts pathway. However, no effect was observed on the uptake of
the hydrocarbon chains within the bilayer. The uptake of SWNTs by SWNT conjugates by the pretreatment of cells with ﬁlipin and nystatin.
the nuclear membrane is attributed to passive ratchet diffusion. Cell (Fig. 5C), All these results suggested that cellular internalization of
mitosis might also play a signiﬁcant role in the internalization of SWNT conjugated with proteins and DNA is through the clathrin-
SNWT-poly (rU) hybrids. During cell division the nuclear envelope dependent endocytosis pathway.
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 641
whether f-CNTs were internalized or not. Even in cases where the
functional groups were electrostatically neutral or negatively charged
in physiological conditions, nanotubes were consistently taken up by
cells. Incubation with cells in the presence of endocytosis inhibitors did
not inﬂuence the cell penetration ability of the f-CNTs. The fact that f-
CNTs were also internalized by a wide variety of cell types, some of
which exhibit deﬁcient phagocytosis (ﬁbroblasts) or lack the machin-
ery for endocytosis (fungi, yeast and bacteria cells), was considered
another indication that the uptake mechanism of the f-CNTs appears to
be passive and endocytosis independent . The cylindrical shape
and high aspect ratio of CNTs allows their penetration through the
plasma membrane, similar to a ‘nanosyringe’. These results are in
contrast to previous reports by Dai et al.  described above. Such
discrepancies were attributed to the sharp differences in the
characteristics of the CNT constructs studied. Macromolecules, such
as nucleic acid, protein, or lipid on CNT could critically change the
interactions between cells and CNTs. The endocytotic mechanism of
uptake is thought to be a result of the macromolecule coat on the CNT
surface recognized by the cells instead of the CNT backbone.
The main internalization pathways to accomplish cellular delivery
of nucleic acids by CNTs were summarized as follows ( Scheme 4
): (1) Endocytosis (D) of nucleic acids that electrostatically
complex with, covalently link to or physically wrap around CNTs
[47,51,86,89,91,99,101,102,105]; (2) Phagocytosis (A) of nucleic acids
[83,106], this mechanism was also demonstrated by the intrinsic
infrared ﬂuorescence of SWNTs with Pluronic F108 surfactant coated
SWNTs ; (3) Penetrating or piercing (C) of nucleic acids adsorbed
on the surface of CNT or complexed with CNT by electrostatic forces
[79,80]. It is noted that this mechanism was fully examined with the
functionalized CNTs [78,80,103], but not with the DNA-CNT com-
plexes [78,80,103]. (4) Injection of nucleic acid through transient
nanochannels (B) formed by CNT under microwave and magnetic
ﬁeld [95–98]. The mechanisms suggested to describe intracellular
release of the nucleic acid from the CNT are as follows: electrostatic
dissociation [78–80,82,83,86,89], enzymatic cleavage of the disulﬁde
linkage that held the nucleic acid onto the CNT [91,92], and nucleic
acid release through excitation of the CNT with NIR radiation .
3.3. CNTs Impact on DNA Conformation and Second Conformational
DNA is not only a one-dimensional string of sequences; it can exist
in many different transient three-dimensional shapes, which can
control their special biological functions. Facilitating or inhabiting
formation of some of the transient structures may offer unique ways
to treat diseases, such as cancer. Recently, Rohs et al. also found that
the shape of DNA, and not just its sequence, offers DNA-binding
proteins the needed directions to ﬁnd their binding sites among the 3
billion base pairs of the human genome. In this section, the inﬂuence
of carbon nanotubes on the structure of nucleic acid and their
conformational changes will be discussed.
Fig. 5. (A) Flow cell cytometry data obtained after incubation in protein–SWNT SWNTs can strongly interact with DNA, both natural DNA [107–
solutions for untreated cells, cells pretreated with 0.45 m sucrose, and K+-depleted 109] and short, custom synthesized oligonucleotides [93,94], and the
medium, respectively. (B) A confocal image that shows cellular uptake of transferring interaction induces DNA helically wrapping around SWNTs. The
protein in HL-60 cells at 37○C. The inset shows the lack of uptake of the transferrin nucleotide bases interact with the nanotube walls via π stacking while
protein after pretreatment of cells in sucrose. (C) Flow cytometry data of cells after
incubation in cholera-toxin B (black bars) and BSA–SWNT (gray bars) for HeLa cells
the phosphate backbone is exposed to water [93,108]. This property
without any pretreatment (control) and cells pretreated with ﬁlipin and nystatin, has been exploited to disperse SWNTs into water solution. One
respectively. The ﬁgure was reproduced from Ref.  with permission. simulation study demonstrated that the carbon nanotube periodically
arranged to ﬁt into the major groove of double stranded DNA .
Very recently, Kostarelos et al.  systematically studied the DNA on SWNTs is surprising ordered. Atomic force microscopy (AFM)
internalization mechanism of CNT functionalized with a series of small phase images shows clearly ss-DNA helically wrapped around
molecules with various types of functional groups ( Scheme 3). They nanotubes [94,108]. Campbell et al.  found that the wrapped
demonstrated that all the functionalized carbon nanotubes (f-CNTs) DNA strands are closely arranged end-to-end in a single layer along
exhibit a capacity to be taken up by a wide range of cells and intra- the SWNTs and the formed periodic pattern is independent to the
cellularly trafﬁc through different cellular barriers. Interestingly, the length and sequence of the wrapping DNA. Classical all-atom
nature of the functional group on the CNT surface did not determine molecular dynamics (MD) simulations were applied to study the
642 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
Scheme 3. (1), Ammonium-functionalized CNT; (2), Acetamido-functionalized CNT; (3), CNT functionalized with ﬂuorescein isothiocyanate (FITC); (4), CNT bifunctionalized with
ammonium groups and FITC; (5), CNT bifunctionalized with methotrexate (MTX) and FITC; (6), shortened CNT bifunctionalized with amphotericin B (AmB) and FITC; (7), shortened
CNT bifunctionalized with ammonium groups and FITC (through an amide linkage). The ﬁgure was reproduced from Ref.  with permission.
self-assembly mechanism and the structure of DNA on SWNTs . bind with the positively charged SWNTs. In contrast, binding of the A-
MD results demonstrated that SWNT induces conformational change DNA onto a positively charged SWNT may promote slightly the A to B
of ssDNA that enables ssDNA to self-assemble via π- π stacking conversion. This is accomplished by the backbone of DNA adsorbing to
interaction between ssDNA bases and SWNT sidewalls. ssDNA is the SWNT, which provides an additional driving force for the
observed to spontaneously wrap around SWNT into compact right- or elongation. Cathcart et al.  extensively studied the time
left-handed helices within a few nanoseconds. The helical wrapping is dependence of a natural salmon tests DNA wrapping on SWNT. They
driven by electrostatic and torsional interactions within the sugar- found that the fraction of DNA bound to the SWNTs increases with
phosphate backbone, which results in ssDNA wrapping around the time, and a complete coating of DNA on the walls of the nanotubes over
SWNTs from the 3’ end to the 5'end. The driving forces for ssDNA a three-month period was observed. High resolution transmission
adsorption and helix formation are both independent of the speciﬁc electron microscopy (HRTEM) images clearly demonstrated the
base sequence, general ssDNA sequence are thus expected to wrap progressive formation. The changes in the SWNT's optical properties
SWNTs in a similar manner, which was experimentally demonstrated were found to coincide with the time at which a full monolayer of DNA
in the work by Gigliotti et al. . Recently Zhao et al.  reported coated the SWNTs. The rate of DNA wrapping was investigated with
their simulation results for the interactions of dsDNA segment in an respect to the sample temperature. The time required for a complete
aqueous solution with a SWNT. Simulations show that DNA binds to DNA monolayer to form on the SWNTs is controlled by a rate-limiting
the external surface of an uncharged or positively charged SWNT on a process with an activation enthalpy of 41 kJ mol-1 (0.43 eV). This low
time scale of a few hundred picoseconds. The hydrophobic end groups energy barrier is attributed to the ﬁnal important step in the wrapping
of DNA are attracted to the hydrophobic SWNT surface of uncharged mechanism, which involves the transformation of the disordered
SWNTs, while the hydrophilic backbone of DNA does not bind to the population of DNA at the surface into a tightly bound array approx-
uncharged SWNT. The adsorption process appears to have negligible imating a monolayer coating. A highly ordered helical wrapping of the
effect on the structure of the B-form DNA segment, but signiﬁcantly DNA around the SWNTs is clearly demonstrated in high resolution
affects the A to B form conversion of A-DNA. The adsorption of A-DNA transmission electron microscope (HRTEM) images (Fig. 6).
onto an uncharged SWNT inhibits the complete relaxation of A-DNA to Ever since O'Connell et al.  ﬁrst observed that individual
B-DNA over a time scale of 3 ns. This is because both ends of A-DNA SWNTs displayed NIR photoluminescence ( NIR PL), there have been a
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 643
Scheme 4. Suggested strategies for cellular delivery of nucleic acids by CNT: phagocytosis of nucleic acids covalently linked to CNT (A), injection of nucleic acids through CNT
nanochannels (B), penetration of nucleic acids adsorbed on the surface of CNT (CI) or complexed with CNT by electrostatic forces (CII); and endocytosis of nucleic acids
electrostatically complexed (DI), covalently linked (DII) or adsorbed (DIII) to CNT. Suggested strategies for intracellular release of the nucleic acid from the CNT: electrostatic
dissociation (E), enzymatic cleavage (F) and NIR radiation (G). The ﬁgure was reproduced from ref.  with permission.
number of reported bioapplications that make use of this convenient the higher order structures of GC-DNA and calf thymus (ct) DNA
property. This distinctive NIR PL originates from their electronic band became unstable upon exposure to the COOH-modiﬁed SWNTs. A
gap of SWNTs, which is sensitive to the local dielectric environment decrease in the Tm melting temperature of 40 °C for GC-DNA was seen
around the SWNTs and yet remains stable to permanent photo- and SWNT binding speciﬁcity was ranked as follows: GC-DNA N ct-
bleaching. Strano et al. [100,115] investigated the conformational DNA N AT-DNA based on AFM and CD (circular dichroism) evidence to
polymorphism of DNA physically wrapped around SWNTs using the support the UV melt proﬁles for each. By examining the CD spectra
native NIR ﬂuorescence of SWNTs. Similar to the dsDNA in solution, more closely, it was found that GC-DNA transformed from the B to A
upon exposure to heavy metal ions, the dsDNA on the SWNTs can also go DNA conformation when the SWNTs were added as a result of their
reversible B-to-Z transition which requires a double stranded helix to binding afﬁnity for the major groove.
separate, change helicity, and re-form, as a process of nucleation and Along a similar vein, Qu et al. found that the COOH-modiﬁed
propagation in series (Fig. 7). The transition of DNA adsorbed on the SWNTs can selectively stabilize human telemetric i-motif DNA under
SWNT or in solution appears to be thermodynamically identical, except physiological conditions or even at pH 8.0  ( Scheme 5). The
the propagation length is much shorter than the DNA in solution. Some negatively charged carboxyl groups on the carbon nanotubes directly
DNA may detach from the SWNT surface during this process. Using a stabilized i-motif CC+ base pairs by providing favorable electrostatic
similar method, they studied the hybridization and hybridization attractions. In addition, a slight conformational change induced the
kinetics of ssDNA physically adsorbed on SWNTs [116,117]. However, TAA loop to be more exposed to the solvent, therefore making it more
they found that hybridization is much slower than the free DNA in sensitive to S1 nuclease cleavage . Consequently, the SWNTs can
solution, with t1/2 = 3.4 h, compared to the free DNA value of t1/2 = be utilized as a sort of catalyst to accelerate the S1 nuclease cleavage
4 min. Although the long-term goal of this team is to develop resilient rate. The enzyme turnover number, kcat, which is the catalytic rate
optical sensors based on CNT for in vitro and in vivo applications, these constant, was 40 s-1 with just i-motif DNA and 885 s-1 with i-motif
results have signiﬁcant implications for DNA and CNTs as therapeutic DNA/SWNT. The reaction rate increased 22-fold. However, the COOH-
nanomedicine. It also gave cautions to the researchers on what might modiﬁed SWNTs can inhibit DNA duplex association as shown by
happen if SWNTs were used as a DNA delivery vehicle: the existence of competitive gel mobility shift assay, CD melting experiments, and
SWNTs would slow down DNA hybridization, destabilize duplex and ﬂuorescence resonance energy transfer (FRET) studies.
triplex DNA, and induce a sequence dependent DNA B-A transition by All message-RNA (mRNA) molecules in eukaryotic cells have a
binding to the DNA major groove . polyadenylic acid [poly(rA)] tail of about 200-250 bases (ca. 70-90 in
Qu and his colleagues  investigated the binding effects of yeast) at the 3’ end. Poly (rA) of mRNA is a critical cellular event in the
SWNTs on DNA self-assembly. They found the SWNTs can destabilize maturation of all eukaryotic mRNAs and it can be catalyzed by an
duplex and triplex DNA and induce a sequence dependent DNA B-A enzyme poly(rA) polymerase (PAP), which is overexpressed in human
transition by binding to the major grooves of DNA. When exposing cancer cells. Thus, molecules capable of recognizing and binding to the
carboxyl-modiﬁed SWNTs to DNA species of varying base pair poly(rA) tail of mRNA might interfere with the full processing of mRNA
compositions, condensations occurred. Speciﬁcally, DNA that by PAP and switch off protein synthesis, representing a new class of
contained greater portions of GC base pairs were easier to condense, potential therapeutic agents. In another continued work by Qu et al.
with the major grooves of GC-DNA serving as the best binding sites for , they uncovered that carboxyl-, or hydroxyl-modiﬁed SWNTs
the COOH-modiﬁed SWNTs as proven by ﬂuorescence competitive can facilitate the self-structuring of single-stranded RNA poly (rA) to
binding assay with DNA intercalators. This is because GC-rich regions form an A A+ duplex-like structure. Using UV melting proﬁles, they
can be dehydrated to a greater extent than other regions. Observing saw that upon passing its melting temperature of 43.5 °C, poly(rA)
the UV melting proﬁles of several types of DNA strands revealed that generated a sigmoidal curve indicative of the same cooperative
644 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
like shape, hydrophobic surface, and their electrical properties.
2). Capability to achieve spatially- and temporally- controlled release
for targeted gene silencing due to their strong adsorption in NIR range.
3). Inﬂuence on conformation and conformational transition of DNA/
siRNA due to their unique shape, modiﬁable surface chemistry, and
their remarkable ﬂexibility. 4). Capability to timely monitoring the
therapeutic effects of DNA/siRNA due to their extremely stable and
strong Raman signal and NIR ﬂuorescence emission.
4. Challenges and Opportunities
4.1. In vivo targeted delivery
Using CNT-mediated gene delivery and treatment for in vivo studies
is still in its infancy. All the reported in vivo studies were administrated
by local intratumoral injection. Targeted delivery of DNA/siRNA to
speciﬁc disease sites can greatly enhance therapeutic efﬁciency and
eliminate side effects, while there is no such effort found in literature for
in vivo targeted delivery of DNA/siRNA medicated by CNT .
Systematic studies on the stability, blood circulation, and pharmacoki-
netics of DNA-CNT conjugates or complexes are urgently needed. In vivo
delivery of chemical anticancer drugs by carbon nanotubes , and
targeted delivery of carbon nanotubes alone  to tumor sites have
been reported. The studies of pharmacokinetics, blood circulation, and
biodistribution of CNT/drug conjugations have demonstrated the
powerful detection and imaging capabilities of CNTs
[41,47,50,77,92,123–127]. However, the combination of the remarkable
NIR optical properties, as well as the imaging and detection capabilities
of CNTs in gene delivery has not been reported yet.
4.2. In vivo permeability and circulation behavior of CNT/DNA/siRNA
Many studies have been performed to investigate how efﬁciently
carbon nanotubes (with or without complexes containing DNA/siRNA)
cross cell membranes and induce the cell death in in vitro cultured cell
Fig. 6. Various HRTEM images showing DNA wrapping of nanotubes in a 32 day old models, or how efﬁciently the carbon nanotubes go to the target organs
sample. The ﬁgure was reproduced from Ref.  with permission. through systemic injection in whole animal models. Although the
cultured cell model allows us to elucidate the detailed uptake of carbon
nanotube DNA/siRNA complexes by cells, it does not tell us how good
binding seen with duplex DNA. This occurred only when the SWNTs the tissue uptake is when delivered through the systemic circulation,
were functionalized with either a carboxyl or hydroxyl group. No such which is the crucial step in the practical use of the carbon nanotube
curve type was observed when analyzing the melting behavior of RNA DNA/siRNA complexes. In contrast, the whole animal study tells us the
by itself, SWNTs by themselves, or with SWNT-functionalized with bio-distribution of the carbon nanotubes (without DNA/siRNA) in
amino groups. It was known that poly(rA) adopts a parallel strand different organs, it tells us the ﬁnal destiny of the carbon nanotubes
double helix conformation as a result of A·A+ protonated adenine without telling us the conditions during the transport, such as the
residues. So the carboxyl or hydroxyl groups on the carbon nanotubes driving force (e.g., CNT concentration difference between that in the
may promote protonation of adenine residues by lowering their pKa lumen of the blood vessel and that in the tissue, and the transmural
values, the favorable interaction with the carboxyl or hydroxyl groups pressure), the permeability properties of different types of microvessels
on the carbon nanotubes facilitated the formation of A A+ duplex-like (e.g., leaky or tight microvessels, under physiological or pathological
structure at neutral pH solutions. conditions), etc. Systematic studies on these kinds of in vivo perme-
ability, stability, blood circulation, and pharmacokinetics of CNT-DNA/
3.4. Summary siRNA-conjugates or complexes are urgently needed.
It is well known that after intravenous injection, micrometer-sized
Gene therapy, especially the recent development in small inference rigid spheroids are cleared immediately in the ﬁrst pass through the
RNA brings great hopes to cure some untreatable diseases. However, microvasculature of various bodily organs. Recently it was reported that
the main issue in gene therapy is to deliver these therapeutic nucleic micelle ﬁlaments of 8 micrometer can be circulated in vivo mainly due to
acids to the targeted sites without eliciting toxicity. CNTs, due to their their ﬂexibility and softness . Due to the elongated shape and small
large surface area, needle like shape, and a series of amazing electronic diameter, huge surface area, and the remarkable ﬂexibility of carbon
and optical properties, are expected to solve the aforementioned nanotubes, it is anticipated that carbon nanotube mediated gene
problems and develop a revolutionary delivery vehicle for gene delivery can pass through not only the leaky tissues, but also the tight
therapy. Compared to the traditional delivery vehicles, the major vascular endothelial barriers with high payload and high-afﬁnity to the
advantaged provided by carbon nanotubes are following: 1). Remark- targeted sites, thus overcoming the challenges facing the traditional
able capability to penetrate into cells, including the difﬁcult transfect- nanocarrier approaches and chemical conjugation approaches as
ing primary-immune cells and bacteria, mainly due to their needle discussed in Section 2.1 and 2.2 of the review.
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 645
Fig. 7. (A) Concentration-dependent ﬂuorescence response of the DNA-encapsulated (6,5) nanotube to divalent chloride counterions. The inset shows the (6,5) ﬂuorescence band at starting
(blue) and ﬁnal (pink) concentrations of Hg2+. (B) Fluorescence energy of DNA-SWNTs inside a dialysis membrane upon removal of Hg2+ during a period of 7 hours by dialysis. (C) Circular
dichroism spectra of unbound d(GT)15 DNA at various concentrations of Hg2+. (D) DNA-SWNT emission energy plotted versus Hg2+ concentration (red curve) and the ellipticity of the 285-nm
peak obtained via circular dichroism measurements upon addition of mercuric chloride to the same oligonucleotide (black curve). Arrows point to the axis used for the corresponding curve.
(E) Illustration of DNA undergoing a conformational transition from the B form (top) to the Z form (bottom) on a carbon nanotube. The ﬁgure was reproduced from Ref.  with permission.
646 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
Scheme 5. Quadruplex “building blocks” and duplex equilibrium. (A) The G-quartet. (B) The C·C+ hemiprotonated base pair of the “building blocks” for quadruplex formation. (C)
Duplex equilibrium shifted by SWNTs. The ﬁgure was reproduced from Ref.  with permission.
4.3. Impact of CNTs on the conformation and conformation transition of 4.4. Toxicity studies of carbon nanotubes with well-deﬁned and well
DNA/siRNA for enhanced therapeutic effects characterized structures
Several studies have already demonstrated the inﬂuence of CNTs Undoubtedly, CNTs are emerging as innovative medicine, which
on the conformation of DNA and RNA [118,119,121]. This property may bring revolutionary strategies to solve some current untreatable
may impact the biological function of the delivered DNA/RNA and/or diseases and reveal untouchable fundamental biological issues.
other DNA/RNA that existed in the cells nearby. For example, the However, their potential toxic effects have become an issue of strong
oligonucleotide molecules wrapped around SWNTs still have the concern for the environment and for health. Such biomedical
capability of hybridizing and undergoing comformational transitions applications will not be realized if there is no proper assessment of
[100,116,117], but the kinetics are largely slowed down compared to the potential hazards of CNTs to humans and other biological systems.
the molecules free in solution. This is very different from the case Tremendous amounts of work have been published on the toxicity of
reported by Mirkin et al.  in which they developed oligonucle- CNTs [101,130–139]. However, the published data are inconsistent
otide modiﬁed gold nanoparticles for intracellular gene regulation. and are widely disputed [77,124,126,127,132,136–138,140–148].
The oligonucleotide molecules were chemically conjugated on the There is a broad agreement that the large diversity of toxicity results
gold nanoparticle surface via a thiol-Au bond. They found that the found in literatures stems from the application of tubes with a wide
oligonucleotides have afﬁnity constants for complementary nucleic range distribution of tube diameters, lengths, and chiralities produced
acids that are higher than the unmodiﬁed oligonucleotide counter- by the current synthesis methods. These disparities may also be due to
parts due to the unusual cooperative binding capability on the nano- tubes with different functionalization, the degree of functionalization,
particles. Compared to the free oligonucleotides, the oligonucleotides and the method of functionalization. Deﬁnite discrimination on the
on the gold nanoparticles are less susceptible to degradation by toxicity of CNTs will continue to be impossible without implemen-
nuclease activity. They attributed this unusual stability to the tight tation of precise measurements, complete characterizations, and the
packing of the antisense oligonucleotide on the Au nanoparticle use of well-deﬁned materials. Furthermore, the application of
surface, which causes steric inhibition of nuclease degradation. different protocols, cell lines, and animal models in evaluating the
However, studies like this that optimize the arrangement of DNA on toxicity and long term fate of the tubes may be very important reasons
carbon nanotubes with an aim to synergistically induce therapeutic for the inconsistency. Standard experimental protocols (including but
effects and minimize unfavorable side effects are largely lacking. not limited to animal models, cell assays, quantiﬁcation, and
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 647
characterization methodologies) should be established so that studies  J.-C. Charlier, X. Blase, S. Roche, Electronic and transport properties of nanotubes,
Rev. Mod. Phys. 79 (2) (2007) 677–732.
may be compared across laboratories. We should be cautious when  25J.W.G Wilder, L.C. Venema, A.G. Rinzler, R.E. Smalley, C. Dekker, Electronic
applying traditional toxicology assays to the safety assessment of structure of atomically resolved carbon nanotubes, Nature 391 (1997) 59–62.
nanoscale materials such as carbon nanotubes . Finally, carefully  R.A. Hatton, A.J. Miller, S.R.P. Silva, Carbon Nanotubes: A Multi-Functional
Materials for Organic Optoelectronics, J. Mater. Chem. 18 (2008) 1183–1192.
thought out long-term studies on the absorption, deposition, metab-  H.J Dai, A. Javey, E. Pop, D. Mann, W. Kim, Y. Lu, Electrical Transport Properties
olism, and excretion (ADME) of CNTs in animals are urgently needed. and Field Effect Transistors of Carbon Nanotubes, NANO: Brief Reports and
Only after all these problems have been solved, one can possibly Reviews 1 (2006() 1–4.
 X. Zhou, J.-Y. Park, S. Huang, J. Liu, P.L. McEuen, Band Structure, Phonon
establish Good Manufacturing Practices (GMP) procedures for clinical Scattering, and the Performance Limit of Single-Walled Carbon Nanotube
use as well as for large scale production of drugs by pharmaceutical Transistors, Phys. Rev. Lett. 95 (2005) 146805.
industries.  T. Durkop, S.A. Getty, E. Cobas, M.S. Fuhrer, Extraordinary Mobility in
Semiconducting Carbon Nanotubes, Nano Lett. 4 (2004) 35–39.
 S Li, Z. Yu, S.-F. Yen, W.C. Tang, P.J. Burke, Carbon Nanotube Transistor Operation
at 2.6 GHz, Nano Lett 4 (2004) 753–756.
 S. Hong, S. Myung, Nanotube Electronics: A ﬂexible approach to mobility, Nat.
Nanotech. 2 (2007) 207–208.
This material is based upon work supported by the National  S.J. Kang, C. Kocabas, T. Ozel, M. Shim, N. Pimparkar, M.A. Alam, S.V. Rotkin, J.A.
Science Foundation under Grant CHE-0750201 and CBET-0933966. He Rogers, High-Performance Electronics Using Dense, Perfectly Aligned Arrays of
Single-Walled Carbon Nanotubes, Nat. Nanotech. 2 (2007) 230–236.
H. also acknowledges the Trustees Research Fellowship Program at  A Kis, A. Zettl, Nanomechanics of carbon nanotubes, Phil. Trans. R. Soc. A 366
Rutgers, The State University of New Jersey. (2008) 1591–1611.
 W. Cheung, P.L. Chiu, R.R. Parajuli, Y. Ma, S.R. Ali, H. He, Fabrication of High
Performance Conducting Polymer Nanocomposites for Biosensors and Flexible
References Electronics: Summary of the Multiple Roles of DNA Dispersed and Functionalized
Single Walled Carbon Nanotubes, J. Mater. Chem. 19 (2009) 6465–6480.
 Y Ma, W. Cheung, D. Wei, A. Bogozi, P.L. Chiu, L. Wang, F. Pontoriero, R.
 Santhakumaran, L.M., A. Chen, C.K.S. Pillai, T. Thomas, H. He, and T.J. Thomas, Mendelsohn, H. He, Improved Conductivity of Carbon Nanotube Networks by
Nanotechnology in Nonviral Gene Delivery in Nanofabrication Towards Biomedical In Situ Polymerization of a Thin Skin of Conducting Polymer, ACS Nano 2 (2008)
Applications: Techniques, Tools, Applications, and Impact, C.S.S.R.H. Kumar, J.; Leuschner, 1197–1204.
C., Editor. 2005, Wiley-VCH.  J. Nijuguna, K. Pielichowski, J.R. Alcock, Epoxy-Based Fibre Reinforced Nano-
 S. Richards, S.-T. Liu, A. Majumdar, J.-L. Liu, R.S. Nairn, M. Bernier, V. Maher, M.M. composites, Adv. Eng. Mater 9 (2007) 835–847.
Seidman, Triplex Targeted Genomic Crosslinks Enter Separable Deletion and  K. Prashantha, J. Soulestin, M.F. Lacrampe, P. Krawczak, Present Status and Key
Base Substitution Pathways, Nucleic Acids Res. 17 (33) (2005) 5382–5393. Challenges of Carbon Nanotubes Reinforced Polyoleﬁns: A Review on Nanocom-
 M.M. Seidman, P.M. Glazer, The potential for gene repair via triple helix formation, J. posites Manufacturing and Performance Issues, Polymer & Polymer Composites
Clin. Invest. 4 (112) (2003) 487–494. 17 (2009) 205–245.
 D.A. Braasch, D.R. Corey, Novel Antisense and Peptide Nucleic Acid Strategies for  B. Lassagne, Y. Tarakanov, J. Kinaret, D. Garcia-Sanchez, A. Bachtold, Coupling
Controlling Gene Expression, Biochemistry 14 (41) (2002) 4503–4510. Mechanics to Charge Transport in Carbon Nanotube Mechanical Resonators,
 A. de Fougerolles, H.-P. Vornlocher, J. Maraganore, J. Lieberman, Interfering with Science 325 (2009) 1107–1110.
Disease: A Progress Report on siRNA-based Therapeutics, Nat. Rev. Drug  P. Kim, L. Shi, A. Majumdar, P.L. McEuen, Thermal Transport Measurements of
Discovery 6 (2007) 443–453. Individual Multiwalled Nanotubes, Phys. Rev. Lett. 87 (2001) 215502.
 D.M. Dykxhoorn, J. Lieberman, The Silent Revolution: RNA Interference as Basic  R.J. Chen, S. Bangsaruntip, K.A. Drouvalakis, N.W.S. Kam, M. Shim, Y.M. Li, W.
Biology, Research Tool and Therapeutic, Annu. Rev. Med. 56 (2005) 401–423. Kim, P.J. Utz, H.J. Dai, Noncovalent functionalization of carbon nanotubes for
 S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Duplexes highly speciﬁ c electronic biosensors, Proc. Nat. Acad. Sci 100 (2003) 4984–4989.
of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells,  P. Cherukuri, S.M. Bachilo, S.H. Litovsky, R.B. Weisman, Near-infrared Fluores-
Nature 411 (2001) 494–498. cence Microscopy of Single-walled Carbon Nanotubes in Phagocytic Cells, J. Am.
 M.A. Lysik, S. Wu-Pong, Innovations in oligonucleotide drug delivery, J. Pharm. Chem. Soc 126 (2004) 15638–15639.
Sci. 92 (8) (2003) 1559–1573.  Z. Liu, X. Sun, N. Nakayama-Ratchford, H.J. Dai, Supramolecular Chemistry on
 S.L. Uprichard, The Therapeutic Potential of RNA Interference, FEBS Lett. 579 Water Soluble Carbon Nanotubes for Drug Loading and Delivery, ACS Nano 1
(2005) 5996–6007. (2007) 50–56.
 B.F. Baker, B.P. Monia, Novel Mechanisms for Antisense-Mediated Regulation of  Z. Liu, S. Tabakman, K. Welsher, H.J. Dai, Carbon Nanotubes in Biology and
Gene Expression, Biochim. Biophys. Acta 1489 (1999) 3–18. Medicine: In vitro and in vivo Detection, Imaging and Drug Delivery, Nano Res 2
 S.T. Crooke, Molecular Mechanisms of Action of Antisense Drugs, Biochim. (2009) 85–120.
Biophys. Acta 1489 (1999) 31–44.  S.J Tans, M.H. Devoret, H.J. Dai, A. Thess, R.E. Smalley, L.J. Geerligs, C. Dekker,
 S.D. Patil, D.G. Rhodes, D.J. Burgess, DNA-Based Therapeutics and DNA Delivery Individual single-wall carbon nanotubes as quantum wires, Nature 386 (1997)
Systems: A Comprehensive Review, AAPS J. 7 (2005) 61–77. 474–477.
 T.V. Achenbach, B. Brunner, K. Heermeier, Oligonucleotide-based Knockdown  P. Chakravarty, R. Marches, N.S. Zimmerman, A.D.E. Swafford, P. Bajaj, I.H.
Technologies: Antisense versus RNA Interference, Chem. Bio. Chem. 4 (2003) Musselman, P. Pantano, R.K. Draper, E.S. Vitetta, hermal ablation of tumor cells
928–935. with anti body-functionalized single-walled carbon nanotubes, Proc. Nat. Acad.
 J.-R Bertrand, M. Pottier, A. Vekris, P. Opolon, A. Maksimenko, C. Malvy, Sci. 105 (2008) 8697–8702.
Comparison of Antisense Oligonucleotides and siRNAs in Cell Culture and in vivo,  Y. Xiao, X. Gao, O. Taratula, S. Treado, A. Urbas, R.D. Holbrook, R.E. Cavicchi, C.T.
Biochem. Biophys. Res. Comm. 296 (2002) 1000–1004. Avedisian, S. Mitra, R. Salva, P.D. Wagner, S. Srivastava, H. He, Anti-HER2 IgY
 G.J Hannon, RNA interference, Nature 418 (2002) 244–251. Antibody-Functionalized Single Walled Carbon Nanotubes for Detection and
 A Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, Potent and Selective Destruction of Breast Cancer Cells, BMC Cancer 9 (2009) 351.
Speciﬁc Genetic Interference by Double-Stranded RNA in Caenorhabditis  N.W.S. Kam, M.J. O'Connell, J. Wickham, H.J. Dai, Carbon Nanotubes as
Elegans, Nature 391 (1998) 806–811. Multifunctional Biological Transporters and Near-Infrared Agents for Selective
 J. Soutschek, A. Akinc, B. Bramlage, C. Charisse, R. Constien, M. Donoghue, S. Cancer Cells Destruction, Proc. Natl. Acad. Sci. 102 (2005) 11600–11605.
Elbashir, A. Geick, P. Hadwiger, J. Harborth, Therapeutic Silencing of an  A. de la Zerda, C. Zavaleta, S. Keren, S. Vaithilingam, S. Bodapati, Z. Liu, J. Levi, T.-J.
Endogenous Gene by Systemic Administration of Modiﬁed siRNAs, Nature 432 Ma, O. Oralkan, Z. Cheng, X. Chen, H.J. Dai, B.T. Khuriyakub, S.S. Gambhir,
(2004) 173–178. Photoacoustic molecular imaging in living mice utilizing targeted carbon
 M.A. Behlke, Progress towards in vivo use of siRNAs, Mol. Ther. 13 (2006) nanotubes, Nat. Nanotech 3 (2008) 557–562.
644–670.  M.J. O'Connell, S.M. Bachilo, C.B. Huffman, V.C. Moore, M.S. Strano, E.H. Haroz, K.L.
 S Akhtar, I.F. Benter, Nonviral Delivery of Synthetic siRNAs in vivo, J. Clin. Invest. Rialon, P.J. Boul, W.H. Noon, W.H. Noon, C. Kittrell, J. Ma, R.H. Hauge, R.B.
117 (2007() 3623–3632. Weisman, R.E. Smalley, Band Gap Fluorescence from Individual Single-Walled
 E. Song, P.C. Zhu, S.-K. Lee, D. Chowdhury, S. Kussman, D.M. Dykxhoorn, Y. Feng, Carbon Nanotubes, Science 297 (2002) 593–596.
D. Palliser, D.B. Weiner, P. Shankar, W.A. Marasco, J. Lieberman, Antibody  K. Welsher, Z. Liu, D. Daranciang, H.J. Dai, Selective Probing and Imaging of Cells
Mediated in vivo Delivery of Small Interfering RNAs via Cell-Surface Receptors, with Single Walled Carbon Nanotubes as Near - Infrared Fluorescent Molecules,
Nature Biotech 23 (2005) 709–717. Nano Lett. 8 (2008) 586–590.
 Y.-L Chiu, A. Ali, C.Y. Chu, H. Cao, T.M. Rana, Visualizing a Correction between  D.A. Heller, S. Baik, T.E. Eurell, M.S. Strano, Single-Walled Carbon Nanotubes
siRNA Localization, Cellular Uptake, and RNAi in Living Cells, Chem. & Biol. 11 Spectroscopy in Live Cells: Towards Long-Term Labels and Optical Sensors, Adv.
(2004) 1165–1175. Mater. 17 (2005) 2793–2799.
 A. Aigner, Delivery Systems for the Direct Application of siRNAs to Induce RNA  A.M. Rao, E. Richter, S. Bandow, B. Chase, P.C. Eklund, K.A. Williams, S. Fang, K.R.
Interference (RNAi) in Vivo, J. Biomed. Biotechnol. 2006 (2006) 1–15. Subbaswamy, M. Menon, A. Thess, R.E. Smalley, G. Dresselhaus, M.S. Dresselhaus,
 P.J.F Harris, Carbon Nanotubes and Related Structures, Cambridge University Diameter-Selective Raman Scattering from Vibrational Modes in Carbon Nano-
Press, 1999. tubes, Science 275 (1997) 187–191.
648 W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649
 K.B. Kostarelos, A. Prato, M. Promises, Facts and Challenges for Carbon Nanotubes  Z. Zhang, X. Yang, Y. Zhang, B. Zeng, S. Wang, T. Zhu, R.B.S. Roden, Y. Chen, Y.
in Imaging and Therapeutics, Nat. Nanotech. 4 (2009) 627–633. Yang, Delivery of Telomerase Reverse Transcriptase Small Interfering RNA in
 D.W. Pack, A.S. Hoffman, S. Pun, P.S. Stayton, Design and Development of Complex with Positively Charged Single Walled Carbon Nanotubes Suppresses
Polymers for Gene Delivery, Nat. Rev. Drug Discovery 4 (2005) 581–593. Tumor Growth, Clin. Cancer Res. 12 (2006) 4933–4939.
 D.J. Gary, N. Puri, Y.-Y. Won, J. Controlled Release, Polymer-based siRNA  R Yang, X. Yang, Z. Zhang, Y. Zhang, S. Wang, Z. Cai, Y. Jia, Y. Ma, C. Zheng, Y. Lu, R.
Delivery: Perspectives on the Fundamental and Phenomenological Distinctions Roden, Y. Chen, Single-Walled Carbon Nanotubes-Mediated in vivo and in vitro
from Polymer-based DNA Delivery 121 (2007) 64–73. Delivery of siRNA into Antigen-Present Cells, Gene Ther. 13 (2006) 1714–1723.
 R. Juliano, M.R. Alam, V. Dixit, H. Kang, Survey and Summary: Mechanisms  X. Yang, Z. Zhang, Z. Liu, Y. Ma, R. Yang, Y. Chen, Multi-Functionalized Single-
and Strategies for Effective Delivery of Antisense and siRNA Oligonucleotides, Walled Carbon Nanotubes as Tumor Cell Targeting Biological Transporters, J.
Nucleic Acids Res. 36 (2008) 4158–4171. Nanoparticle Res. 10 (2008) 815–822.
 M. Banan, N. Puri, The ins and outs of RNAi in Mammalian Cells, Curr. Pharm.  Y. Liu, Z.-L. Yu, Y.-M. Zhang, D.-S. Guo, Y.-P. Liu, Supramolecular Architectures of
Biotechnol. 5 (2004) 441–450. Cyclodextrin-Modiﬁed Chitosan and Pyrene Derivatives Mediated by Carbon
 S.M. Elbashir, J. Martinez, A. Patkaniowska, W. Lendeckel, T. Tuschl, Functional Nanotubes and Their DNA Condensation, J. Am. Chem. Soc. 31 (2008) 10431–10439.
Anatomy of siRNAs for Mediating Efﬁcient RNAi in Drosophila Melanogaster  Y. Liu, D.-C. Wu, W.-D. Zhang, X. Jiang, C.-N. He, T.S. Chung, S.H. Goh, K.W. Leong,
Embryo Lysate, Embo J. 20 (2001) 6877–6888. Polyethylenimine-Grafed Multiwalled Carbon Nanotubes for Secure Noncova-
 N. Bessis, F.J. GarciaCozar, M.C. Boissier, Immune Responses to Gene Therapy lent Immobilization and Efﬁcient Deliveyr of DNA 44 (2005) 4782–4785.
Vectors: Inﬂuence on Vector Function and Effector mechanisms, Gene Ther. 11  O. Boussif, F. Lezoualc'h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, J.P.
(2004) S10–S17. Behr, A versatile vector for gene and oligonucleotide transfer into cells in culture
 D.D. Paula, V.M.L.B. Bentley, R.I. Mahato, Hydrophobization and bioconjugation and in vivo: polyethylenimine, Proc. Natl. Acad. Sci. 92 (1995) 7297.
for enhanced siRNA delivery and targeting, RNA 13 (2007) 431–456.  M. Ogris, P. Steinlein, M. Kursa, K. Mechtler, R. Kircheis, E. Wagner, The size of
 A Aigner, Applications of RNA Interference: Current State and Prospects for DNA/transferrin-PEI complexes is an important factor for gene expression in
siRNA-based Strategies in vivo, Appl. Microbiol. Biotechnol. 76 (2007) 9–21. cultured cells, Gene Ther. 5 (1998() 1425.
 C Kimchi-Sarfaty, S. Brittain, S. Garﬁeld, N.J. Caplen, Q. Tang, M.M. Gottesman,  N Jia, Q. Lian, H. Shen, C. Wang, X. Li, Z. Yang, Intracellular Delivery of Quantum
Efﬁcient Delivery of RNAi Effectors via in vitro-Packaged SV40 Pseudovirions, Dots Tagged Antisense Oligodeoxynucleotides by Functionalized Multiwalled
Hum. Gene Ther. 16 (2005) 1110–1115. Carbon Nanotubes, Nano Lett 7 (2007) 2976–2980.
 S Zhang, B. Zhao, H. Jiang, B. Wang, B. Ma, Cationic Lipids and Polymers Mediated  R Krajcik, A. Jung, A. Hirsch, W. Neuhuber, O. Zolk, Functionalization of Carbon
Vectors for Delivery of siRNA, J. Control Release 123 (2007) 1–10. Nanotubes Enable Non-covalent Binding and Intracellular Delivery of Small
 A.M. Chen, L.M. Santhakumaran, S.K. Nai, P.S. Amenta, T. Thomas, H. He, T.J. Interfering RNA for Efﬁcient Knock-down of Genes, Biochem. Biophys. Res.
Thomas, Oligodeoxynucleotide Nanostructure Formation in the Presence of Comm. 369 (2008) 595–602.
Polypropyleneimine Dendrimers and Their Uptake in Breast Cancer Cells,  N.W.S. Kam, Z. Liu, H.J. Dai, Functionalization of Carbon Nanotubes via Cleavable
Nanotechnology 17 (2006) 5449–5460. Disulﬁde Bonds for Efﬁcient Intracellular Delivery of siRNA and Potent Gene
 M.L. Patil, M. Zhang, O. Taratula, O.B. Garbuzenko, H. He, T. Minko, Internally Silencing, J. Am. Chem. Soc. 127 (2005) 12492–12493.
Cationic Polyamidoamine (PAMAM-OH Dendrimer for siRNA Delivery: Effet of  Z Liu, M. Winters, M. Holodniy, H.J. Dai, siRNA Delivery into Human T Cells and
the Degree of Quaternization and Cancer Targeting, Biomacromolecules 10 Primary Cells with Carbon Nanotube Transporters, Angew. Chem. Int. Ed. 46
(2009) 258–266. (2007) 2023–2027.
 Taratula, O., O.B. Garbuzenko, P. Kirkpatrick, I. Pandya, R. Savla, V.P. Pozharov, H.  M. Zheng, A. Jagota, E.D. Semke, B.A. Diner, R.S. Mclean, S.R. Lustig, R.E.
He, and T. Minko, Surface-Engineered Targeted PPI Dendrimers for Efﬁcient Richardson, N.G. Tassi, DNA-assisted Dipersion and Separation of Carbon
Intracellular and Intratumoral siRNA delivery. J. Control Release 2009(In Press). Nanotubes, Nature Materials 2 (2003) 338–342.
 M.L. Patil, M. Zhang, S. Betigeri, O. Taratula, H. He, T. Minko, Surface-Modiﬁed  M. Zheng, A. Jagota, M.S. Strano, A.P. Santos, P. Barone, S.G. Chou, B.A. Diner, M.S.
and Internally Cationic Polyamidoamine Dendrimers for Efﬁcient siRNA Dresselhaus, R.S. Mclean, G.B. Onoa, G.G. Samsonidze, E.D. Semke, M. Usrey, D.J.
Delivery, Bioconjugate Chem. 19 (2008) 1396–1403. Walls, Structure-Based Carbon Nanotube Sorting by Sequence-Dependent DNA
 D.B. Kirpotin, D.C. Drummond, Y. Shao, M.R. Shalaby, K. Hong, U.B. Nielsen, J.D. Assembly, Science 302 (2003) 1545–1548.
Marks, C.C. Benz, J.W. Park, Antibody Targeting of Long-Circulating Lipidic  J. Rojas-Chapana, M.A. Correa-Duarte, Z.F. Ren, K. Kempa, M. Giersig, Enhanced
Nanoparticles Does Not Increase Tumor Localization but Does Increase Introduction of Gold Nanoparticles into Vital Acidothiobacillus Ferrooxidans by
Internalization in Animal Models, Cancer Res. 66 (2006) 6732–6740. Carbon Nanotube-Based Microwave Electroporation, Nano Lett. 4 (2004) 985–988.
 M. Manoharan, RNA Interference and Chemically Modiﬁed Small Interfering  J. Rojas-Chapana, J. Troszczynska, I. Firkowska, C. Morsczeck, M. Giersig, Multi
RNAs, Curr. Opin. Chem. Biol. 8 (2004) 570–579. Walled Carbon Nanotubes for Plasmid Delivery in Escherichia Coli Cells, Lab on a
 D.A. Braasch, S. Jensen, Y. Liu, K. Kaur, K. Arar, M.A. White, D.R. Corey, RNA Chip 5 (2005) 536–539.
interference in mammalian cells by chemically modiﬁed RNA, Biochemistry 42  D. Cai, J.M. Mataraza, Z.-H. Qin, Z. Huang, T.C. Chiles, D. Carnahan, K. Kempa, Z.F.
(2003) 7967–7975. Ren, Highly Efﬁcient Molecular Delivery into Mammalian Cells Using Carbon
 Y.L. Chiu, T.M. Rana, siRNA Function in RNAi: A Chemical Modiﬁcation Analysis, Nanotube Spearing, Nature Methods 2 (2005) 449–454.
RNA 9 (2003) 1034–1048.  D. Cai, C.A. Doughty, T.B. Potocky, F.J. Dufort, Z. Huang, D. Blair, K. Kempa, Z.F. Ren, T.C.
 M. Amarzguioui, T. Holen, E. Babaie, H. Prydz, Tolerance for Mutations and Chiles, Carbon Nanotube-Mediated Delivery of Nucleic Acids Does Not Result in Non-
Chemical Modiﬁcations in a siRNA, Nucleic Acids Res. 31 (2003) 589–595. speciﬁc Activation of B Lymphocytes, Nanotechnology 18 (2007) 365101.
 F. Czauderna, M. Fechtner, S. Dames, H. Aygun, A. Klippel, G.J. Pronk, K. Giese, J.  N.W.S Kam, Z. Liu, H.J. Dai, Carbon Nanotubes as Intracellular Transporters for
Kaufmann, Structural Variations and Stabilising Modiﬁcations of Synthetic Proteins and DNA: An Investigation of the Uptake Mechanism and Pathway,
siRNAs in Mammalian Cells, Nucleic Acids Res. 31 (2003) 2705–2716. Angew. Chem. Int. Ed. 45 (2006) 577–581.
 T.P. Prakash, C.R. Allerson, P. Dande, T.A. Vickers, N. Siouﬁ, R. Jarres, B.F. Baker,  D.A. Heller, E.S. Jeng, T.K. Yeung, B.M. Martinex, A.E. Moll, J.B. Gastala, M.S. Strano,
E.E. Swayze, R.H. Griffey, B. Bhat, Positional effect of chemical modiﬁcations on Optical Detection of DNA Conformational Polymorphism on Single Walled
short interference RNA activity in mammalian cells, J. Med. Chem. 48 (2005) Carbon Nanotubes, Science 311 (2006) 508–511.
4247–4253.  M.L. Becker, J.A. Fagan, N.D. Gallant, B.J. Bauer, V. Bajpai, E.K. Hobbie, S.H. Lacerda,
 J. Elmen, H. Thonberg, K. Ljungberg, M. Frieden, M. Westergaard, Y. Xu, B. Wahren, K.B. Migler, J.P. Jakupciak, Length-Dependent Uptake of DNA-Wrapped Single-
L. Zicai, T. Koch, C. Wahlestedt, Locked nucleic acid (LNA) mediated improve- Walled Carbon Nanotubes, Adv. Mater 19 (2007) 939–949.
ments in siRNA stability and functionality, Nucleic Acids Res. 33 (2005) 439–447.  Q. Lu, J.M. Moore, G. Huang, A.S. Mount, A.M. Rao, L.L. Larcom, P.C. Ke, RNA
 D.B Rozema, D.L. Lewis, D.H. Wakeﬁeld, S.C. Wong, J.J. Klein, P.L. Roesch, S.L. Polymer Translocation with Single Walled Carbon Nanotubes, Nano Lett 4 (2004)
Bertin, T.W. Reppen, Q. Chu, A.V. Blokhin, J.E. Hagstrom, J.A. Wolff, Dynamic 2473–2477.
Polyconjugates for Targeted Deliery of siRNA to Hepatocytes, Proc. Natl. Acad.  K. Kostarelos, L. Lacerda, G. Pastorin, W. Wu, S. Wieckowski, J. Luangsivilay, S.
Sci. 104 (2007) 12982–12987. Godefroy, D. Pantarotto, J.-P. Briand, S. Muller, M. Prato, A. Bianco, Celluar Uptake
 Z. Liu, C. Davis, W. Cai, L. He, X. Chen, H.J. Dai, Circulation and Long - Term Fate of of Functionalized Carbon Nanotubes is Independent of Functional Group and Cell
Functionalized Biocompatible Single-Walled Carbon Nanotubes in Mice Probed Type, Nature Nanotech 2 (2007) 108–113.
by Raman Spectroscopy, Proc. Natl. Acad. Sci. 105 (2008) 1410–1415.  L. Lacerda, A. Bianco, M. Prato, K. Kostarelos, Carbon Nanotube Cell Translocation
 D. Pantarotto, R. Singh, D. McCarthy, M. Erhardt, J.-P. Briand, M. Prato, K. and Delivery of Nucleic Acids in vitro and in vivo, J. Mater. Chem. 18 (2008) 17–22.
Kostarelos, A. Bianco, Functionalized Carbon Nanotubes for Plasmid DNA Gene  H. Jin, D.A. Heller, M.S. Strano, Single-Particle Tracking of Endocytosis and
Delivery, Angew. Chem. 116 (2004) 5354–5358. Exocytosis of Single - Walled Carbon Nanotubes in NIH-3 T3 Cells, Nano Lett.
 R. Singh, D. Pantarotto, D. McCarthy, O. Chaloin, J. Hoebeke, C.D. Partidos, J.-P. 8 (2008) 1577–1585.
Briand, M. Prato, A. Bianco, K. Kostarelos, Binding and Condensation of Plasmid  A.E Porter, M. Gass, K. Muller, J.N. Skepper, P.A. Midgley, M. Welland, Direct
DNA onto Functionalized Carbon Nanotubes: Toward the Construction of Imaging of Single-Walled Carbon Nanotubes in Cells, Nature Nanotech. 2 (2007)
Nanotube-Based Gene Delivery Vectors, J. Am. Chem. Soc. 127 (2005) 4388–4396. 713–717.
 A. Bianco, J. Hoebeke, S. Godefroy, O. Chaloin, D. Pantarotto, J.-P. Briand, S. Muller,  N Nakashima, S. Okuzono, H. Murakami, T. Nakai, K. Yoshikawa, DNA Dissolves
M. Prato, C.D. Partidos, Cationic Carbon Nanotubes Bind to CpG Oligodeoxynu- Single-walled Carbon Nanotubes in Water, Chem. Lett. 32 (2003) 456–457.
cleotides and Enhance Their Immunostimulatory Properties, J. Am. Chem. Soc.  B Gigliotti, B. Sakizzie, D.S. Bethune, R.M. Shelby, J.N. Cha, Sequence-Independent
127 (2004) 58–59. Helical Wrapping of Single-Walled Carbon Nanotubes by Long Genomic DNA,
 X. Wang, J. Ren, X. Qu, Targeted RNA Interference of Cyclin Mediated by Nano Lett 6 (2006) 159–164.
Functionalized Single-Walled Carbon Nanotubes Induces Proliferation Arrest  H. Cathcart, S.J. Quinn, V. Nicolosi, J.M. Kelly, W.J. Blau, J.N. Coleman,
and Apoptosis in Chronic Myelogenous Leukemia K562 Cells, ChemMedChem 3 Spontaneous Debundling of Single-Walled Carbon Nanotubes in DNA-Based
(2008) 940–945. Dispersions, J. Phys. Chem. C 111 (2007) 66–74.
W. Cheung et al. / Advanced Drug Delivery Reviews 62 (2010) 633–649 649
 G. Lu, P. Maragakis, E. Kaxiras, Carbon Nanotube Interaction with DNA, Nano Lett.  L. Zhu, D.W. Chang, L. Dai, Y. Hong, DNA Damage Induced by Multiwalled Carbon
5 (2005) 897–900. Nanotubes in Mouse Embryonic Stem Cells, Nano Lett. 7 (2007) 3592–3597.
 J.F. Campbell, I. Tessmer, H.H. Thorp, D.A. Erie, Atomic Force Microscopy Studies  M.L. Schipper, N. Nakayama-Ratchford, C.R. Davis, N.W.S. Kam, P. Chu, Z. Liu, X.
of DNA-Wrapped Carbon Nanotube Structure and Binding to Quantum Dots, J. Sun, H.J. Dai, S.S. Gambhir, A Pilot Toxicology Study of Single-Walled Carbon
Am. Chem. Soc. 130 (2008) 10648–10655. Nanotubes in a Small Sample of Mice, Nature Nanotech. 3 (2008) 216–221.
 R.R. Johnson, A.T.C. Johnson, M.L. Klein, Probing the Structure of DNA-Carbon  V.E. Kagan, Y.Y. Tyurina, V.A. Tyurin, N.V. Konduru, A.I. Potapovich, A.N. Osipov,
Nanotube Hybrids with Molecular Dynamics, Nano Lett 8 (2008) 69–75. E.R. Kisin, D. Schwegler-Berry, R. Mercer, V. Castranova, A. Shvedova, Direct and
 X. Zhao, J.K. Johnson, Simulation of Adsorption of DNA on Carbon Nanotubes, J. Indirect Effects of Single Walled Carbon Nanotubes on RAW 264.7 Macrophages:
Am. Chem. Soc. 129 (2007) 10438–10445. Role of Iron, Toxicol. Lett. 165 (2006) 88–100.
 H Cathcart, V. Nicolosi, J.M. Hughes, W.J. Blau, J.M. Kelly, S.J. Quinn, J.N. Coleman,  C.-C Chou, H.-Y. Hsiao, Q.-S. Hong, C.H. Chen, Y.-W. Peng, H.-W. Chen, P.C. Yang,
Ordered DNA Wrapping Switches on Luminescence in Single-Walled Nanotube Single-Walled Carbon Nanotubes Can Induce Pulmonary Injury in Mouse Model,
Dispersions, J. Am. Chem. Soc. 130 (2008) 12734–12744. Nano Lett. 8 (2008) 437–445.
 H. Jin, E.S. Jeng, D.A. Heller, P.V. Jena, R. Kirmse, J. Langowski, M.S. Strano,  C.A. Poland, R. Dufﬁn, I. Kinloch, A. Maynard, W.A. Wallace, A. Seaton, V. Stone, S.
Divalent Ion and Thermally Induced DNA Conformational Polymorphism on Brown, W. Macnee, K. Donaldson, Carbon Nanotubes Introduced into the Abdominal
Single-Walled Carbon Nanotubes, Macromolecules 40 (2007) 6731–6739. Cavity of Mice Show Asbestos-like Pathogenicity in a Pilot Study, Nature Nanotech. 3
 E.S. Jeng, P.W. Barone, J.D. Nelson, M.S. Strano, Hybridization Kinetics and (2008) 423–428.
Thermodynamics of DNA Adsorbed to Individually Dispersed Single-Walled  Y. Sato, A. Yokoyama, K. Shibata, Y. Akimoto, S. Ogino, Y. Nodasaka, T. Kohgo, K.
Carbon Nanotubes, Smal 3 (2007) 1602–1609. Tamura, T. Akasaka, M. Uo, K. Motomiya, B. Jeyadevan, M. Ishiguro, R. Hatakeyama,
 E.S. Jeng, A.E. Moll, A.C. Roy, J.B. Gastala, M.S. Strano, Detection of DNA F. Watari, K. Tohji, Inﬂuence of Length on Cytotoxicity of Multi-Walled Carbon
Hybridization Using the Near-Infrared Band-Gap Fluorescence of Single Walled Nanotubes against Human Acute Monocytic Leukemia Cell line THP-1 in vitro and
Carbon Nanotubes, Nano Lett 6 (2006) 371–375. Subcutaneous Tissue of Rats in vivo, Mol. BioSyst. 1 (2005) 176–182.
 X. Li, Y. Peng, J. Ren, X. Qu, Carboxyl-modiﬁed Single-walled Carbon Nanotubes  C.M. Sayes, F. Liang, J.L. Hudsona, J. Mendeza, W. Guo, J.M. Beach, V.C. Moorea, C.D.
Selectively Induce Human Telomeric i-motif Formation, Proc. Natl. Acad. Sci. 103 Doyle, J.L. West, W.E. Billups, K.D. Ausman, V.L. Colvin, Functionalization Density
(2006) 19658–19663. Dependence of Single-Walled Carbon Nanotubes Cytotoxicity in vitro, Toxicol.
 X. Li, Y. Peng, X. Qu, Carbon nanotubes Selective Destabilization of Duplex and Lett. 161 (2006) 135–142.
Triplex DNA and Inducing B-A Transition in Solution, Nucleic Acids Res. 34  H. Dumortier, S. Lacotte, P. Pastorin, R. Marega, W. Wu, D. Bonifazi, J.P. Briand, M.
(2006) 3670–3676. Prato, S. Muller, A. Bianco, Functionalized Carbon Nanotubes Are Non-Cytotoxic
 Y. Peng, X. Li, J. Ren, X. Qu, Single-walled Carbon Nanotubes Binding to Human and Preserve the Functionality of Primary Immune Cells, Nano Lett 6 (2006)
Telomeric i-motif DNA: Signiﬁcant Acceleration of S1 Nuclease Cleavage Rate, 1522–1528.
Chem. Commun. 48 (2007) 5176–5178.  Z. Li, T. Hulderman, R. Salmen, R. Chapman, S.S. Leonard, S.H. Young, A. Shvedova, M.
 C. Zhao, Y. Peng, Y. Song, J. Ren, X. Qu, Self-Assembly of Single-Stranded RNA on I. Luster, P.P. Simeonova, 377-382, Cardiovascular Effects of Pulmonary Exposure to
Carbon Nanotube: Polyadenylic Acid to Form a Duplex Structure, Small 4 (2008) Single-Wall Carbon Nanotubes, Environ. Health Perspect. 115 (2007) 377–382.
656–661.  Y. Zhu, W.X. Li, Cytotoxicity of Carbon Nanotubes, Sci. China Ser. B-Chem 51 (11)
 J.E. Podesta, K.T. Al-Jamal, M.A. Herrero, B. Tian, H. Ali-Boucetta, V. Hegde, A. Bianco, (2008) 1021–1029.
M. Prato, K. Kostarelos, Antitumor Activity and Prolonged Survival by Carbon-  Warheit, D.B., B.R. Laurence, K.L. Reed, D.H. Roach, G.A.M. Reynolds, and T.R. Webb,
Nanotube-Mediated Therapeutic siRNA Silencing in a Human Lung Xenograft Comparative Pulmonary Toxicity Assessment of Single Wall Carbon Nanotubes in Rats.
Model, Small 5 (2009) 1176–1185. Toxicol. Sci., 2003: p. In press.
 Z. Liu, K. Chen, C. Davis, S. Sherlock, Q. Cao, X. Chen, H.J. Dai, Drug Delivery with  G. Jia, H. Wang, L. Yan, X. Wang, R. Pei, T. Yan, Y. Zhao, X. Guo, Cytotoxicity of Carbon
Carbon Nanotubes for In vivo Cancer Treatment, Cancer Res. 68 (2008) 6652–6659. Nanomaterials: Single-Wall Nanotube, Multi-Wall Nanotube, and Fullerene,
 Z Liu, W. Cai, L. He, N. Nakayama, K. Chen, X. Sun, X. Chen, H.J. Dai, In vivo Environ. Sci. Technol. 39 (2005() 1378.
Biodistribution and Highly Efﬁcient Tumor Targeting of Carbon Nanotubes in  J.C Carreor-Sanchez, A.L. Elias, R. Mancilla, G. Arrellin, H. Terrones, J.P. Laclette, M.
Mice, Nature Nanotech. 2 (2007) 47–52. Terrones, Biocompatibility and Toxicological Studies of Carbon Nanotubes
 P.W. Barone, M.S. Strano, Reversible Control of Carbon Nanotube Aggregation for a Doped with Nitrogen, Nano Lett 6 (2006) 1609.
Glucose Afﬁnity Sensor. Angew, Chem. Int. Ed. 45 (2006) 8138–8141.  C.-W. Lam, J.T. James, R. McCluskey, S. Arepali, R.L. Hunter, A Review of Carbon
 R. Singh, D. Pantarotto, L. Lacerda, G. Pastorin, C. Klumpp, M. Prato, A. Bianco, K. Nanotube Toxicity and Assessment of Potential Occupational and Environmental
Kostarelos, Tissue Biodistribution and Blood Clearance Rates of Intravenously Health Risks, Crit. Rev. Toxicol. 36 (2006) 189.
Administrated Carbon Nanotube Radiotracers, Proc. Natl. Acad. Sci. 103 (2006)  A. Magrez, S. Kasas, V. Salicio, N. Pasquier, J.W. Seo, M. Celio, S. Catsicas, B.
3357–3362. Schwaller, L. Forro, Functionalized Carbon Nanotubes Are Non-Cytotoxic and
 P. Cherukuri, C.J. Gannon, T.K. Leeuw, Mammalian Pharmacokinetics of Carbon Preserve the Functionality of Primary Immune Cells, Nano Lett. 6 (2006) 1522.
Nanotubes Using Intrinsic Near-Infrared Fluorescence, Proc. Natl. Acad. Sci. 103  S.K. Smart, A.I. Cassady, G.Q. Lu, D.J. Martin, The Biocompatibility of Carbon
(2006) 18882–18886. Nanotubes, Carbon 44 (6) (2006) 1034–1047.
 Y. Geng, P. Dalhaimer, S. Cai, R. Tsai, M. Tewari, T. Minko, D.E. Discher, Shape  D.B. Warheit, What is Currently Known about the Health Risks Related to Carbon
effects of ﬁlaments versus spherical particles in ﬂow and drug delivery, Nature Nanotube Exposures? Carbon 44 (6) (2006) 1064–1069.
Nanotech. 2 (2007) 249–255.  J.M. Worle-Knirsch, K. Pulskamp, H.F. Krup, Oops They Did It Again! Carbon
 N.L Rosi, D.A. Giljohann, C.S. Thaxton, A.K.R. Lytton-Jean, M.S. Han, C.A. Mirkin, Nanotubes Hoax Scientists in Viability Assays, Nano Lett 6 (2006) 1261–1268.
Oligonucleotide-Modiﬁed Gold Nanoparticles for Intracellular Gene Regulation,  R.M. Reilly, Carbon Nanotubes: Potential Beneﬁts and Risks of Nanotechnology in
Science 312 (2006) 1027–1030. Nuclear Medicine, J. Nucl. Med. 48 (7) (2007) 1039–1042.