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DNA and carbon nanotubes as medicine

VIEWS: 216 PAGES: 17

									                                                             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
Gene therapy
                                                       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: (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 [1]. 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) [23]. 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 [3]. 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 [24]. 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 [25]. 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 [26].
cytomegalovirus retinitis (Vitravene) and nearly twenty others in late-          Carbon nanotubes have excellent electronic properties: metallic
stage clinical trials [12]. 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 [27]. 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 [28],
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 [15]. 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 [33]. The strength results from the
specifically induced silencing of homologous genes [16]. 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) [38]. Furthermore, the heat transmis-
mammalian immune response and facilitating gene-specific silencing                sion capacity of individual CNTs at room temperature has been shown to
[7]. 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 [39]. 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 [5] 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. [148].
                                                       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. [24] 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 [55].
    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 [56]. 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 [57]. It makes the RNA backbone more susceptible to
onto the polyaromatic surface of nanotubes [42]. 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 [58]. The second
ligands [43].                                                                              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 [6]. 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
[44]. 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 [59]. There
wavelengths include the tissue transparent region of the electromag-                       is much interest in developing nonviral gene delivery vehicles [54]
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 [48]. 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 [47].                                                                                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 [46]. 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. [43] 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. [53] also summarized the promises, facts and current                     have demonstrated high transfection efficiency in vitro [55]. 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 [77]. 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 [68], 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. [78].
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 [69].                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 [79]. 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 [6].           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 [76] 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. [78]
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                [85] 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 [86] 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 [80].                      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 [81]. 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 [82], 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 [87]. 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 [88]. 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 [88]. 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. [83]. 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 [91]. The first step in this
by Wang et al. [81]. 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 [84] (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. [84] 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 [47].
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 [92].                  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 [91] 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. [97] proposed a novel spearing                          technique with respect to primary B lymphocytes [98]. 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. [99] 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. [101] 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. [47] 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 [92]. 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. [102], 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 [78]. 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. [99] 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
                                                                                         [104]): (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 [41]; (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 [47].

                                                                                         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. [99] with permission.                   simulation study demonstrated that the carbon nanotube periodically
                                                                                         arranged to fit into the major groove of double stranded DNA [110].
    Very recently, Kostarelos et al. [103] 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. [111] 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. [103] with permission.

self-assembly mechanism and the structure of DNA on SWNTs [112].                        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. [114] 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. [108]. Recently Zhao et al. [113] 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. [49] 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. [104] 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 [118] ( 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 [120]. 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 [118].                                                      polyadenylic acid [poly(rA)] tail of about 200-250 bases (ca. 70-90 in
    Qu and his colleagues [119] 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                      [121], 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 [122].
                                                                                    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 [123], and
                                                                                    targeted delivery of carbon nanotubes alone [124] 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. [114] 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 [128]. 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. [100] 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. [118] 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. [129] 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

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