Prospects for the Use of Carbon Nanotubes in Medicine-Juniper Publishers
Authored
by J Igielska-Kalwat
Abstract
Newly discovered compounds showing unique properties
can have profound effect on development of medicine. As far as carbon is
concerned, a discovery of great consequences for medicine was that of a
new allotropic form of carbon known as fullerene. Recently much
interest has been paid to the application of carbon nanotubes as
carriers of therapeutic drugs, biosensors, in gene therapy or in
anticancer therapy.
Keywords: Nanotubes; Nanotechnology; Medicine
Abbrevations:
SWCNT: Single-Walled Carbon Nanotubes; MWCNT: Multi-Walled Carbon
Nanotubes; CVD: Chemical Vapour Deposition; CNT: Carbon Nanotubes; NIR:
Near-Infrared Radiation; f-CNT: Functionalized Carbon Nanotubes; CNPs:
Carbon Nanotubes Polymers Composites
Introduction
Nanotechnology has become one of the most intensely
developing area of research and it combines the achievements from many
branches of science. In 1985, Harold Kroto, Robert Curl and Richard
Smalley discovered a new allotropic and molecular form of carbon making
icosahedral hollow structures, known as fullerene. As this structure is
hollow inside, it can host metal atoms or molecules of chemical
compounds. It is expected that this form of drug administration may
revolutionise medical industry in near future [1]. Since 1991, so since
the discovery of carbon nanotubes, they have been studied in a number of
research centres of which the pioneering group has been headed by Prof.
SumioIijima, NEC, Japan. New properties and new possibilities of
application are discovered daily. New composites with carbon nanotubes
have been proposed, showing high mechanical strength, high electric
conductivity, and exceptional mechanical or electric features. In
medical therapy the use of carbon nanotubes permits application of
active substances to exactly defined target which shortens the time in
which the drug reaches the target and increases the effectiveness of
therapy.
Structures preparation and basic characterisation
An interesting example of carbon nanostructures are
carbon nanotubes. They are made of graphene sheets wrapped to make
seamless cylinders. The diameter of nanotubes is by about 10 thousand
times smaller than that of a human hair [2,3]. The nanotube obtained by
wrapping a single sheet of graphene is called the single-wall nanotube.
Depending on the mode of
wrapping of the graphene sheet the nanotubes can be chiral and
non-chiral. With respect to the shape of the edge, the non-chiral
nanotubes are divided into armchair and zigzag ones. Nanotubes can end
with the fullerene hemispheres. The diameters of the smallest nanotubes
are of an order of 1 nm. The ratio of the nanotube length to its
diameter can be of an order of 102–103. Depending on the number of
graphene layers forming the structure, carbon nanotubes can be divided
into single-walled carbon nanotubes (SWCNT) and multi-walled carbon
nanotubes (MWCNT), composed of many concentric layers of graphene [4].
Varying the chiral angle between hexagons and the tube axis, SWCNTs can
be either metals or semiconductors, with relatively large (circa 0.5eV
for typical diameter of 1.5nm) or small band gaps (circa 10meV), even if
their diameters are nearly identical [5]. With diameters of 1-2nm, and
lengths ranging from as short as 50nm up to 1cm, SWNTs are
one-dimensional (1-D) nanomaterials which may behave distinctly from
spherical nanoparticles in biological environment [6].
A few methods for production of nanotubes have been
proposed, all of them are based on slow condensation of hot vapour of
carbon atoms. In the process of their production a mixture of different
structures is obtained: single- and multi-walled nanotubes of different
wall configurations, toruses, spirals and fullerenes [3,7]. The methods
for production of carbon nanotubes include: laser-induced graphite
evaporation, electric arc technique, chemical vapour deposition (CVD)
[8]. One of the specific properties of nanotubes is their large surface
area. Moreover, depending on the diameter and degree of twisting
they can behave as a metal or as a semiconductor. Thanks to
very strong bonds between carbon atoms in the graphene layer,
the nanotubes show high mechanical resistance, their Young
modulus is very high, of 1012N/m2, so their deformations
are elastic, they are highly resistant to bending or stretching.
Nanotubes can conduct current of very high density, of an order
of 109A/cm2, as they show very low electric resistance. Their
thermal conductivity reaches 6000W/(K x m) at T = 300K, which
is very useful for removal of heat from electronic elements.
The property very attractive from the medical point of view is
the possibility of regulating the nanotubes biocompatibility by
chemical modifications [9-12].
Functioalization for medical use of nanotubes
Unmodified carbon nanotubes (CNT) are hardly soluble in
water, which restricts their medicinal use. To avoid this problem,
the graphene sheets are functionalised in different ways [13-
16]. In general the methods of CNT functionalization are divided
into endohedral and exohedral ones. In the endohedral methods
the hollow space inside the tubes is filled with substances of
different polarity, e.g. metals or chemical compounds, while
in the exohedral methods the external walls of the tubes are
modified [17,18]. The functionalization of nanotubes is realised
via adsorption of proteins, amino acids, enzymes or nucleic
acids [12,13,19]. It can be also performed by adsorption of
chemical compounds through pyridine rings. Modification with
pyrrolidine rings gives the species soluble in certain organic
solvents [16]. Improvement in nanotubes solubility can also be
achieved by introduction of certain biochemical compounds, e.g.
bovine serum albumin,into a solution of nanostructures [12,20].
Application in medicine
The application of nanotubes in medicine is related to the
possibility of their bio-functionalization and the control of their
biocompatibility [21,22].
Drug delivery
Much promising is the use of nanotubes as carriers
transporting biologically active drugs to certain well-defined
sites. Thanks to their specific properties multi-walled nanotubes
have become the basis of a drug delivery system directly to
a target site. CNT have been used to facilitate the absorption
of amphotericin B, an antifungal antibiotic. At first CNTs are
subjected to carboxyl acid so that the -COOH groups would attach
to the outer surface, then they are subjected to diaminetrethylene
glycol, which permits incorporation of the antibiotic. On the
other hand, CNTs modified with fluorescein isothiocyanate can
be used for imagining [23-25].
Cancer therapy
Recently, the use of carbon nanotubes in photodynamic
therapy has been studied. Curley et al. have injected carbon
nanotubes into the cancer tissue in the rabbit liver and then
irradiated this spot with radio waves, which resulted in damaging
of the cancer cells [21]. CNT can be modified with certain specific
antibodies that would capture some well-defined substances, e.g.
biological growth factors in tumours. Thanks to the use of CNTs it
has become possible to resign from the traditional radiotherapy
with high-energy radiation destroying also healthy tissues
[23,24]. A considerable problem is their strongly hydrophobic
character, responsible for the tendency to aggregation and
for possible difficulties with their removal from the organism.
However, as a result of surface functionalization of CNT e.g. with
silica, their character can change into hydrophilic, so that they
could form a stable dispersion.
The capturing of therapeutic drug inside the nanotubes is just
the first step of goal-directed chemotherapy. They should also be
endowed with the properties allowing their accumulation in the
tumour and ensuring the release of the active drug at this site. It
can be achieved in two ways. The nanotubes can be filled with a
ferromagnetic core thanks to which their movements would be
controlled by a magnetic field or the nanotubes can be equipped
with a cap at the end and this cap would respond to different pH
values [24,25].
Chemotherapeutics delivery systems
Due to toxic side effects of most chemotherapeutic agents,
there are some limitations in their use. Because of that fact, it is
very important to find a method to develop cell-targeting drug
formulations with a wide therapeutic index. Carbon nanotubes
have shown great promise as, conveyance” for targeted drug
delivery [25]. CNT can be applied as carriers of anticancer drugs
to deliver them to a target site, within the so-called goal-directed
therapy [26,27]. Drugs administered in traditional chemotherapy
affect the whole organism and destroy also healthy cells, while
the drugs delivered via goal-directed therapy are released only
after having reached the tumour. Another advantage of this
method is the possibility of using the carefully adjusted dose
of the drug so that to destroy the tumour and not to cause
undesired effects. A CNT with a diameter of 80 nm can hold up
to 5 million drug molecules [28]. One of method of incorporating
drug into CNT is steered molecular dynamic simulation, of which
the general principle is to apply an external force to particles
in a specific direction by use of harmonic restraint in order to
create better change of the particle coordinates. Drugs can either
attach to the outer surface of the CNT via functional groups
through either covalent or noncovalent bonding, including
hydrophobic, Ï€–Ï€ stacking, and electrostatic interactions
(egzohedral modification) or be put inside the CNT (endohedral
modification) [29,30]. Drug-loaded CNT has to recognize its site
of action and the routes by which it can be delivered to target
cells. One of the major techniques used involves coating the
surface of the CNT with a particular antibody having affinity for
the target cancer cell. Another method used in targeted cancer
therapy is modification of the CNT with folic acid and with
photosensitizer from the group of porphyrin [31]. As a result
of using the laser irradiation of the appropriate wavelength on
presented structure, singlet oxygen evolution process can be observed. Singlet oxygen destroys the tumor cells Depending on
the grade of cancer lesions of varying efficacy is achieved, but it
is not less than 60% [32].
Thermal ablation
Specific thermal ablation using single-walled carbon
nanotubes targeted by covalently-coupled monoclonal
antibodies is used to destroy tumor cells [33]. Egzohedral
modification with those antibodies make the system recognize
the cancer cells. The ability of CNTs to absorb near-infrared
(NIR) radiation (wavelength 700–1100 nm) and convert it
into heat gives an opportunity to create a new generation of
structures for cancer photo-therapy. NIR light can effectively
penetrate healthy tissue and ablate any cells to which the
CNTs are attached [34]. To increase therapeutical effect of
thermal ablation, the chemotherapeutic agent and actinium
are placed inside the CNT structure. After introducing modified
nanostructure into the patient’s body, determined body area is
subjected to the laser radiation with near infrared. As a result
of CNT overheating the chemotherapeutic agent is released. It’s
activity is enhanced by actinium radioactivity [35,36]. In addition
to its lethal activity, hyperthermia has been used in the clinical
treatment of solid tumors because of enhancing the efficiency of
chemo- or radiotherapy. The local increase in temperature also
increases the permeability of blood vessels, which can enhance
the delivery of drugs to tumors [37].
T-cell therapy
T cells, called also T lymphocytes are a type of lymphocyte
that plays a significant role in cell-mediated immunity. Their
name is derived from the process of their maturation that takes
place in the thymus (although some also mature in the tonsils).
It was recently found to use tumour specific T cells taken from
a patient’s own blood and use them against tumour targets. A
promising method to reproducibly expand T cells in human body
is by attaching the stimuli for T cells onto artificial substrates
with high surface area. Carbon nanotubes polymers composites
(CNPs) can be used as an artificial antigen-presenting cell to
efficiently expand the number of T lymphocytes. It was proved,
that tumour growth was significantly delayed for those mices
that was adoptively transferred with CNP-cultured T cells in
comparison with those without any treatment at day 14 of
therapy [38].
Biosensors
Another interesting application of nanotubes is in biosensors
that are able to detect specific molecules [39]. For this application
CNTs surface must be functionalized with the enzymes sensitive
to a given substance. In such a way it would be possible to make
a biosensor detecting in a continuous way the level of glucose in
blood. The conductivity of CNT depends on the functionalization
so it will change with subsequent molecules of sugar bound
to the enzyme. The nanometric size of the device permits
placement of such a detector in the organism. In combination
with electro chromic materials this device permits design of an
intelligent lens whose colour would inform about the level of
sugar. After appropriate functionalization CNT can be used for
observation of cell properties and changes taking place in cells
during their development, for control of enzymatic reactions, ion
transportation and secretion of proteins or products of chemical
transformations. Detection of DNA particles and neoplastic cells
in the early stages of growth is possible thanks to a large surface
area of CNT and their ability of electron transportation [40,41].
Biocompatibility
A very important problem related to carbon nanotubes is
their biocompatibility. This problem has been studied by many
research groups. Particularly interesting results have been
reported by Chlopeket al., who tested the influence of CNT in
cells, using osteoblasts and fibroblasts. They tested the effect of
MWCNT modified with polysulphone on the lifetime of the cells
and the amount of secreted collagen. The presence of CNT only to
a small degree weakened the cells’ viability, but it promoted the
amount of secreted collagen. The effect of increased synthesis of
collagen can be used for regeneration of bones and soft tissues
with CNT stimulating their growth [42].
Gene therapy/DNA delivery
Gene therapy is one of much promising methods for the
treatment of cancer and genetic disorders. Genes are transported
by special virus-based or not virus-based carriers, the latter
groups includes liposomes, polymers and nanoparticles. The
use of liposomes brings a risk of undesirable effects such as
immunological reaction, inflammatory states or oncogenesis.
In general the non-virus based carriers not always ensure the
sufficient level of gene expression, which has stimulated the
search for new carriers [43]. The large-molecular and cationic
character of functionalized carbon nanotubes (f-CNT) permits
electrostatic interaction with plasmid DNA. To evaluate the
f-CNT abilities to make complexes with nucleic acids and their
translocation, Pantarotto et al. combined at different rates f-CNT
and plasmid DNA, containing the marker gene of β galactosidase.
TEM images revealed the presence of CNT-DNA complexes. The
functionalised SWCNT were seen in the form of bundles among
them were the plasmids in the form of ring clusters or highly
folded structures. The degree of expression of the marker gene
of β galactosidase confirmed the complexes ability to permeate
inside cells. The level of expression of the gene studied was found
to be from 5 to 10 times higher for the complexes of f-SWCNT
and DNA than for the DNA helix alone [44]. Gene transportation
by carbon nanotubes can be used silencing certain genes. Zhang
et al. have studied the complexes of f-SWCNT and sRNA of the
telomerase gene. They have reported a fast penetration of the
complexes into a certain line of mouse cancer cells, release of
sRNA and effective suppression of the telomerase gene [45].
Toxicity
The toxicity of carbon nanotubes can be related to the
high ratio of tubes lengths to diameters and to the toxicity of
the material of which it is made – graphene. The nanotubes show greater toxicity towards the respiratory system than the
particles of diameters larger than 100 nm. CNT are classified
as nanoparticles that can participate in the unknown and
unpredictable interactions with biological systems [46]. Their
toxicity can be limited by subjecting them to appropriate
functionalization. According to the in vitro studies by Sayeset
al., SWCNT covalently functionalized by sulphophenyl and
carboxyphenyl groups have weaker cytotoxic effect then the
suspension of purified SWCNT in water, stabilised with a
1% solution of surfactant [47]. Because of their size, carbon
nanotubes can be treated as fibrous material showing usually
high toxicity towards the lungs. Lam et al. have studied the
toxicity of SWCNT in mice. They have checked the health risk
of exposure to purified and non-purified CNT. According to the
results, depending on the dose and the content of a catalyst, the
use of SWCNT led to the appearance of granulomas and produced
interstitial inflammations, further pathological changes could
lead to bronchogenic inflammation of the lungs [48]. Shvedowa
et al. studied the effect of SWCNT in different doses on thelarynx
of mice exposed in CNT in the form of aerosol. The results
permitted identification of two SWCNT fractions differing in
the size of particles and toxic effect. The first fraction made of
CNT aggregates was responsible for the appearance of acute
inflammation and formation of granulomas at the site of their
accumulation. The second fraction, made of thin delicate CNT of
diameters smaller than 50 nm, stimulate the process of fibrosis
and contribute to increase in the walls of alveoli in the regions
which the primary aggregates did reach [49]. Administration of
CNT through the trachea and aspiration through the throat lead
to agglomeration of CNT in the upper part of bronchi and to the
beginning of fibrosis. The skin exposure to CNT has been also
studied. Huczkoand Lange have performed a dermatological test
on 40 volunteers and Draize test, which revealed the irritating
effect of CNT on the skin [50]. On the other hand, the study
performed on the lines of human keratinocytes undermine these
results. Shvedovaet al. have studied the effect of non-purified
SWCNT on the line of immortalised human keratinocytes
(HaCaT), and reported on the increase in the oxidation stress
with the simultaneous use of antioxidants, loss of viability and
morphological changes in the structure of the cells. To some
degree the results were related to a relatively high content of
the catalyst (used for the CNT synthesis) residues (~30%) [51].
For this reason the authors emphasised the risk related to direct
contact of the skin to CNTs. As follows from the hitherto studies,
evaluation of the CNT toxicity is not a simple task. Results of such
evaluations are often contradictory. It can be concluded that nonpurified
carbon nanotubes show rather high toxicity related to
the presence of the catalysts (Fe, Ni, Co, Zn) residues. Exposure
to purified CNT, especially in high concentrations, leads to
much weaker toxic effects. The least toxic are the functionalized
nanotubes that are to be used for medical applications [34].
Conclusion
The application of carbon nanotubes can significantly
contribute to solving special problems in medical therapy.
Nanotubes can be used for in vivo production of tissues and for
controlling of their development. Much promising is the use
of carbon nanotubes as carriers of therapeutic drugs in goaldirected
therapy or DNA in gene therapy.
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