Content
1. Chapter (1)
1.1 Introduction
1.2 Carbon-Based Nanomaterials
1.2.1
Fullerenes
Biomedical Applications of Fullerenes
1.2.2
Carbon Nanotubes
Biomedical Applications of Carbon Nanotubes
1.2.3
Graphene and its derivatives
Biomedical Applications of Graphene Oxide
1.2.4
Carbon Quantum Dots
Biomedical Applications of Carbon quantum dots
1.2.5
Nanodiamonds
Biomedical Applications of Nanodiamonds
1.3 Toxicity of Carbon-based Nanomaterials
2. Chapter (2)
2.1.
Carbon nanotubes as nerve tissue reconstructing platforms
2.2.
Carbon nanotubes ability in guiding neuronal cells' behavior
2.2.1. Carbon nanotube-based devices to promote growth and neurite
elongation
2.2.2. Patterned carbon nanotube platforms to spatially direct neuronal
growth
2.2.3. Carbon nanotube growth substrates ability to improve nerve cell
performance
2.3.
Mechanisms underlying carbon nanotube-mediated neuromodulation
2.4.
CNTs in drug delivery
2.4.1. Delivery of drugs for various therapies
2.4.2. Diagnostic tools
2.4.3. Genetic engineering
2.4.4. Artificial implants
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
2.4.5. As catalyst
2.5.
Cellular uptake of carbon nanotubes
2.6.
Limitations and Challenges
2.6.1. Lack of solubility/dispersion
2.6.2. Challenge in reproduction of identical CNTs
2.6.3. High production cost
2.6.4. High energy desirable
2.7.
Conclusion of neuroregeneration and neuroprotection
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Chapter 1
1.
1.1.
Nanomaterials
Introduction
The prefix ‘nano’ is referred to a Greek prefix meaning ‘dwarf’ or something very small and
depicts one thousand millionth of a meter (10−9m). Nanotechnology is one of the most promising
technologies of the 21st century. It is the ability to convert the nanoscience theory to useful
applications by observing, measuring, manipulating, assembling, controlling and manufacturing
matter at the nanometer scale. The National Nanotechnology Initiative (NNI) in the United States
define Nanotechnology as “a science, engineering, and technology conducted at the nanoscale (1
to 100 nm), where unique phenomena enable novel applications in a wide range of fields, from
chemistry, physics and biology, to medicine, engineering and electronics”. This definition
suggests the presence of two conditions for nanotechnology. The first is an issue of scale:
nanotechnology is concerned to use structures by controlling their shape and size at nanometer
scale. The second issue has to do with novelty: nanotechnology must deal with small things in a
way that takes advantage of some properties because of the nanoscale [1].
The scale of dimensions adopted for the applicability of nanotechnology is usually <100 nm. It
was first put forward by the American physicist and Nobel Prize laureate Richard Feynman
introduce the concept of nanotechnology in 1959. During the annual meeting of the American
Physical Society, Feynman presented a lecture entitled “There’s Plenty of Room at the Bottom”
at the California Institute of Technology (Caltech). In this lecture, Feynman made the hypothesis
“Why can’t we write the entire 24 volumes of the Encyclopedia Britannica on the head of a
pin?”, and described a vision of using machines to construct smaller machines and down to the
molecular level [2]. In this lecture, he explored the benefits that might accrue to us if we started
manufacturing things on the very small scale. The ideas he put forward were remarkably
prescient. For example, he foresaw the techniques that could be used to make large-scale
integrated circuits and the revolutionary effects that the use of these circuits would have upon
computing. He talked about making machines for sequencing genes by reading DNA molecules.
He foresaw the use of electron microscopes for writing massive amounts of information in very
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
small areas. He also talked about using mechanical machines to make other machines with
increasing precision and talked about exploiting the interactions of quantized spins, a kind of
‘spin logic’, which is only now being studied. These new ideas demonstrated that Feynman’s
hypotheses have been proven correct, and for these reasons, he is considered the father of
modern nanotechnology. Although he did not use the term “nanotechnology” but it was first
coined by Norio Taniguchi in 1974 [3].
Nanotechnology is hailed as having the potential to increase the efficiency of energy
consumption, help clean the environment, and solve major health problems. It is said to be able
to massively increase manufacturing production at significantly reduced costs. Products of
nanotechnology will be smaller, cheaper, lighter yet more functional and require less energy and
fewer raw materials to manufacture, claim nanotech advocates.
Nanotechnology has several applications such as:
1) Nanotechnology in Drugs; Nanotechnology employs curative agents at the nanoscale
level to develop nanomedicine. The field of biomedicine comprising nanobiotechnology,
drug delivery, biosensors, and tissue engineering has been powered by nanoparticles [4].
As nanoparticles comprise materials designed at the atomic or molecular level, they are
usually small sized nanospheres [5]. Hence, they can move more freely in the human
body as compared to bigger materials. Nanoscale sized particles exhibit unique
structural, chemical, mechanical, magnetic, electrical, and biological properties.
Nanostructures stay in the blood circulatory system for a prolonged period and enable the
release of amalgamated drugs as per the specified dose. Thus, they cause fewer plasma
fluctuations with reduced adverse effects [6]. Being nano-sized, these structures penetrate
in the tissue system, facilitate easy uptake of the drug by cells, permit an efficient drug
delivery, and ensure action at the targeted location. The uptake of nanostructures by cells
is much higher than that of large particles with size ranging between 1 and 10 µm [4, 7].
Hence, they directly interact to treat the diseased cells with improved efficiency and
reduced or negligible side effects.
2) Nanotechnology in Diagnostic Techniques; Nanowires may be laid down across a
microfluidic channel (Figure 1), and as particles flow through the microfluidic channel,
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
the nanowire sensors pick up the molecular signatures of these particles and relay the
information to a signal analyzer. Such systems can detect the presence of altered genes
associated with the disease and can help researchers pinpoint the position of these genetic
changes [8]. Zheng et al. reported the preparation of a silicon nanowire (SiNW)
biosensor array for the simultaneous detection of multiple cancer biomarkers in a single
versatile detection platform [9]. The real-time detection of three cancer markers
(prostate-specific antigen, carcinoembryonic antigen, and mucin-1) using SiNW
biosensors functionalized with three cognate antibodies was demonstrated [10]. The
simultaneous high-sensitivity analysis of multiple biomarkers could further facilitate the
early detection of cancer [11,12].
Figure 1. Schematic diagram of the SiNW biosensor for label-free detection of
carbohydrate-protein interactions
3) Nanotechnology in Textiles; a new frontier in clothing technology is nanoengineered
functional textiles. The advantage of nanomaterials concerns creating function without
altering the comfort properties of the substrate [13]. Textile is a universal interface and
ideal substrate for the integration of nanomaterials, electronics, and optical devices.
These engineered materials should seamlessly integrate into garments, and be flexible
and comfortable while having no allergic reaction to the body. Additionally, such
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
materials need to satisfy weight, performance, and appearance properties (color). A
significant challenge in the textile industry is that conventional approaches to
functionalize fabrics do not lead to permanent effects.
decreases imparted functional effects.
For example, laundering
Hence, nanotechnology can play a part to
introduce new and permanent functions to fabrics. Textiles can be nanoengineered to
have specific functions including hydrophobicity, antibacterial properties, conductivity,
antiwrinkle properties, antistatic behavior, and light guidance and scattering (Figure 2).
Using nanotechnology, these properties can be achieved without affecting breathability
or texture. Such materials may be in the form of surface coatings, voided patterns, fillers,
or foams.
Figure 2. A diagrammatic representation of various utilizations of
nanotechnology-based textiles.
4) Nanotechnology in agricultural and food production; Nanobiotechnology provided
industry with new tools to modify genes and even produce new organisms. This is due
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
to the fact that it enables nanoparticles, nanofibers, and nanocapsules to carry foreign
DNA and chemicals that modify genes [14]. In addition, novel plant varieties may be
developed using synthetic biology (a new branch that draws on the techniques of
genetic engineering, nanotechnology, and informatics). In a recent breakthrough in this
area, researchers completely replaced the genetic material of one bacterium with that
from another – transforming it from one species to another [15]. Nanotechnology
possesses the potential to augment agricultural productivity through genetic
improvement of plants and animals along with cellular level delivery of genes and drug
molecules to specific sites in plants and animals.
Agri-food
nanotechnology
is
multidisciplinary
in
nature
(Figure
3).
Nanotechnology application to the agriculture and food sectors is relatively recent
compared with its use in drug delivery and pharmaceuticals [16]. Nanotechnology has
the potential to protect plants, monitor plant growth, detect plant and animal diseases,
increase global food production, enhance food quality, and reduce waste for “sustainable
intensification”. Food and agricultural production are among the most important fields of
nanotechnology application [17].
Figure 3. Multidisciplinary nature of agri-food nanotechnology.
5) Nano-fertilizers; Fertilizers based on nanotechnology have the potential to surpass
conventional fertilizers. In nanofertilizers, nutrients can be encapsulated by
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
nanomaterials, coated with a thin protective film, or delivered as emulsions or
nanoparticles [18]. Nano-based slow-release or CR fertilizers have the potential to
increase the efficiency of nutrient uptake. Engineered nanoparticles are useful for
mitigating the chronic problem of moisture retention in arid soils and enhancing crop
production by increasing the availability of nutrients in the rhizosphere [19]. Coating and
binding of nano- and subnano-composites help to regulate the release of nutrients from
the fertilizer capsule.
6) Nano-bioelectronics; nano-bioelectronics represents a rapidly expanding interdisciplinary
field that combines nanomaterials and nanoscience with biology and electronics, and in
so doing, offers the potentials to overcome existing challenges in bioelectronics and open
up new frontiers. For example, an affinity-based biosensor, such as a protein or DNA
sensor, utilizes a surface-immobilized recognition probe to selectively interact with the
biological analyte in solution and yields a electrical signal directly proportional to analyte
concentration [20]. In addition, bioelectronic devices interfaced to electrogenic cells,
such as neurons or cardiomyocytes, can record and/or stimulate bioelectrical activities in
the cells or corresponding tissues (e.g., brain, heart or muscle), by interconverting ionic
and electronic currents at the device/cell interface [21].
Despite these advantages, nanotechnology has some pitfalls such as; nano-particles can get into
the body through the skin, lungs and digestive system, thus creating free radicals that can cause
cell damage or mass poisoning or unwanted neurological effects. Once nano-particles are in the
bloodstream, they will be able to cross the blood-brain barrier (BBB). The most dangerous nanoapplication use for military purposes is the Nano-bomb that contain engineered self multiplying
deadly viruses that can continue to wipe out a community, country or even a civilization.
Nanobots because of their replicating behavior can be big threat for GREY GOO, Grey Goo is a
hypothetical end-of-the-world scenario involving molecular nanotechnology in which out-ofcontrol self-replicating robots consume all matter on Earth while building more of themselves-a
scenario known as ecophagy (“eating the environment”). The ability to alter substances at a
molecular level is a powerful skill and, left in the wrong hands, could lead to misuse [22].
1.2.
Carbon-Based Nanomaterials
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
In the last few decades, carbon-based nanomaterials (CBN) have shown tremendous impact in
the biomedical field with their ability to deliver therapeutic molecules and allow visualization of
cells and tissues, which are necessary for the cure and treatment of diseased and damaged
tissues. CBN, as members of the carbon family, include fullerenes, carbon nanotubes (CNTs),
graphene (G) and its derivatives (graphene oxide (GO)), nanodiamonds (NDs), and carbon-based
quantum dots (CQDs). The possible biomedical applications of CBN, as depicted in (Figure 4),
include bioimaging, fluorescence labelling of cells, stem cell engineering, biosensing, drug/gene
delivery, and photothermal and photodynamic therapy.
Figure 4. Carbon-based nanomaterials (CBNs) and their diverse applications in
theranostics. Optical imaging; reproduced with permission from the American
Chemical Society. Raman imaging; reproduced with permission from the
American Chemical Society. Photodynamic photothermal therapy; reproduced
with permission from the John Wiley and Sons.
The unique optical properties of CBN, i.e., intrinsic fluorescence, tunable narrow emission
spectrum, and high photostability, allow their potential use in the imaging and diagnosis of cells
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
and tissues. Furthermore, modification of their surfaces with functional groups (carboxylic acid,
hydroxyl, and epoxy) allows the opportunity to optimize their properties. Besides excellent
optical properties, CBN possess high surface areas and mechanical and electrical properties,
which make them one of the most desirable and qualified candidates for theranostic applications.
Above all, the biological safety of CBN, which is related to their aqueous stability and
interactions with cells and tissues, is one of the fundamental issues for their practical biomedical
application.
Some recent studies have spurred their potential utility in anticancer and anti-inflammatory
treatments. For example, CBN stimulate reactive oxygen species (ROS) when taken up by cancer
cells, leading to lipid and DNA damage, and cell death. Also, graphene materials affect the
metabolic activity of cancer-diseased macrophages, increasing the ROS levels and damaging the
mitochondrial membrane, which cause cell death by apoptosis.
Although carbon-based molecules such as fullerenes (C60, C70, and C84) were discovered in 1985,
the existence of C60 was predicted in 1970. Most fullerenes (e.g., C60) are spheroid in shape,
although oblong shapes like a rugby ball also exist (e.g., C70). Later, the discovery of the carbon
nanotube (CNT) in 1991 boosted the research in the field of carbon related nanomaterials.
Structurally, CNT is a one-atom-thick sheet of graphite rolled into a tube with a diameter of one
nanometer, which exhibits different properties depending solely on how the nanotubes are rolled.
The properties of CNTs include high tensile strength and modulus, making them very stiff. Also,
due to their one-dimensional nano-tubular structure, their electrical and thermal conductivity
greatly increase and even surpass that of conductive metals. Recently, the discovery of a one
atom layer thick atomic carbon sheet, i.e., graphene, has placed CBN at the forefront in the
materials science world. Some of the notable characteristics of graphene are its high intrinsic
carrier mobility (200000 cm2 V-1 s-1), thermal conductivity (~5000 W m-1 K-1), Young’s modulus
(~1.0 TPa), and optical transmittance (~97.7%). CBN have been explored in various fields,
including the electronics and semiconductor industry, data storage devices, sensors and
bioelectronics, composite materials, energy research, catalysis, and most recently biomedicine
and theranostics [23].
1.2.1. Fullerenes
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Buckminsterfullerene (C60), which was discovered in 1985, has received significant attention due
to its unique photophysical and photochemical properties. Buckminsterfullerene falls into the
category of spherical fullerenes. The key feature of fullerenes is their ability to act as sensitizers
for the photoproduction of singlet oxygen (1 O2) ROS, and thus are utilized for blood
sterilization and photodynamic cancer therapy. However, the dispersibility of fullerenes is a
major issue for their use in nanomedicine. Their low solubility in many solvents, especially in
water, where singlet oxygen has a long lifetime, is the main problem. Thus, several methods
have been developed to functionalize fullerenes with hydrophilic groups to enhance their water
solubility. Consequently, the developed fullerenes have found potential use as antimicrobial,
antiviral, and antioxidant agents.
The antioxidant role of fullerenes, i.e., scavenging free radicals including ROS and reactive
nitrogen species (RNS), has spurred their biomedical applications. Glutathione C60 derivatives
help protect cells from nitric oxide-mediated apoptotic death. When pre-incubated with C60 the
IgE dependent mediator released in human mast cells (hMCs) and peripheral blood basophils
was significantly inhibited, demonstrating the role of fullerenes as a negative regulator of allergic
response.
Figure 5. Buckminsterfullerene has sixty carbon atoms joined
by covalent bonds
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Fullerenes are also potential photosensitizers. They can absorb photons in the ultraviolet and
visible electromagnetic spectrum to produce photo-excited fullerene species in the triplet state
and lead to the generation of singlet oxygen or ROS depending on the polarity of the medium.
Furthermore, light-harvesting antennae can be attached to fullerenes to increase the quantum
yield of ROS production. Therefore, fullerenes can be used in photodynamic therapy (PDT) for
treating cancer and killing microorganisms.
The nanoscale cage-like structure of fullerenes allows the construction of molecular or
particulate entities, where one or more functional groups are covalently attached to the fullerene
cage surface in a geometrically controlled manner. This is applicable for the targeted delivery of
drugs across biological membranes and receptor ligands for agonizing or antagonizing cellular
and enzymatic processes. Liposome formulation provides an alternative route to prepare
fullerenes for pharmaceutical applications with enhanced distribution, absorption and delivery
efficiency.
Substantial scientific knowledge on fullerene medicine has been gained; however, the progress in
clinical studies is lacking due to the concerns regarding long-term safety and toxicity of
fullerenes. In contrast, fullerene-based cosmetic products have been clinically tested and used in
human skincare for many years, suggesting that at least the topical application of fullerenes is
safe. Additionally, the stable cage-like structure of fullerenes provides an abundant room for the
encapsulation of atoms, molecules, and ions. For example, water-soluble gadolinium
metallofullerenes (gadofullerenes) are very promising magnetic resonance imaging (MRI)
contrast agents due to their high relaxivity [24].
1.2.1.1.
I.
Biomedical Applications of Fullerenes
Functionalized fullerenes as drug-delivery nanoparticles
Paclitaxel-embedded buckysomes (PEBs) are spherical nanostructures in the order of 100–200
nm composed of the amphiphilic fullerene (AF-1), AF-1 embedding the anti-cancer drug
paclitaxel inside its hydrophobic pockets. Similar to Abraxane®, the US Food and Drug
Administration (FDA)-approved drug for treating diseases such as metastatic breast cancer, our
water-soluble fullerene derivatives enable the uptake of paclitaxel without the need for non11
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
aqueous solvents, which can cause patient discomfort and other unwanted side effects. However,
our preliminary studies indicate that PEBs might be capable of delivering even higher amounts
of paclitaxel than those delivered via Abraxane®. By delivering an increased amount of
paclitaxel, we can hope to reduce infusion times and expect higher tumor uptake, resulting in a
greater anticancer efficacy. Another attractive feature of our fullerene-based delivery vectors is
that their nanoscale dimensions favor passive targeting, which enables them to accumulate at
tumor sites by entering through leaky vasculature present in the endothelial cells of the tumor
tissue. Additionally, the fullerene moiety can be easily functionalized to attach targeting agents,
which facilitate active targeting. PEBs also provide an easy feature of adding targeting groups to
their fullerene moieties. In PEBs, both liposomal and nanoparticle technologies are combined to
create nanostructures that function as novel drug carriers. This approach is advantageous because
it may improve circulation times in the blood, shields the anticancer drug against enzymatic
degradation and reduces uptake by the reticuloendothelial system (RES). The size of the PEBs is
designed to be less than 200 nm to avoid RES uptake. The presence of dendritic groups on the
outside of the PEBs can also provide stealth function to reduce clearance
II.
Reactive oxygen species (ROS) quenching by functionalized fullerenes
Ever since Krusic and colleagues documented the potential of fullerenes to scavenge ROS, there
has been a great interest in using fullerenes as an antioxidant. However, it is important to
remember that while functionalizing fullerenes to make them water soluble, the free radical
scavenging properties must be maintained. In 1997, Dugan and colleagues published a pathbreaking article on “carboxyfullerenes as neuroprotective agents.”
They suggested that C60 derivatives might constitute antioxidant compounds useful in biological
systems. Carboxyfullerenes were efficient against excitotoxic necrosis and provided protection
against two forms of neuronal apoptosis. This led to the idea that oxidative stress is a critical
downstream mediator in disparate necrotic and apoptotic neuronal deaths. The study also showed
that amphiphilicity is a desirable feature in the functionalization, increasing intercalation into
brain membranes and neuroprotective efficacy. The article demonstrated that C60 derivatives can
indeed function as neuroprotective drugs in vivo. In another study, Lin and colleagues presented
in vitro data demonstrating that carboxyfullerenes possess an antioxidative property and is
12
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
capable of suppressing iron-induced lipid peroxidation. Their in vivo study showed
neuroprotection by carboxyfullerene against iron-induced degeneration of the nigrostriatal
dopaminergic system. Also, they reported that the intranigral infusion of carboxyfullerene
appeared to be nontoxic to the nigrostriatal dopaminergic system of rats. Other research studies
that followed, confirmed the protective activity of carboxyfullerenes against oxidative stress and
their potential as a free radical scavenger [25].
1.2.2. Carbon Nanotubes
Carbon nanotubes (CNTs) are rolled up seamless cylinders of graphene sheets with unique
intrinsic properties (Figure 6). Based on the number of graphene layers in the cylindrical tubes,
CNTs are classified as single wall carbon nanotubes (SWCNTs) and multiwall carbon nanotubes
(MWCNTs). The diameter of SWCNTs and MWCNTs varies from 0.4 to 2.5 nm and a few
nanometers to 100 nm, respectively. Each layer in MWCNTs interacts through van der Waals
forces and a variety of combinations of 2D crystals with different electrical, optical and
mechanical properties is possible to constitute multilayered CNTs to provide different physical
phenomena and device functionality.
Figure 6. Graphene sheets are rolled up forming carbon nanotubes.
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
However, the poor dispersibility of CNTs is one of the biggest barriers for their use in
nanomedicine. Thus, several functionalization routes have been developed to disperse them and
consequently improve their biocompatibility. Covalent functionalization is possible through the
defective carbon atoms on the sidewall or at the end, where carboxylic acid groups or
carboxylated fractions are generated through oxidization, which are then chemically modified via
amination or esterification. Recently, several polymers, metals, and biological molecules have
been used to graft to the surface of carboxylated CNTs.
Nevertheless, concerns for the biocompatibility (cell and tissue toxicity) of CNTs have been
raised as a practical issue, and many in-depth studies are still ongoing, although it is generally
accepted that when the surface of CNTs is properly functionalized (modified), their cell and
tissue compatibility can be improved significantly. Functionalized CNTs have offered great
opportunities in many biomedical applications, including biosensing, disease diagnosis and
treatment. They are used to detect various biological targets, allow biomedical imaging, and
deliver therapeutic molecules including drugs and genes.
Their intrinsic spectroscopic properties, including Raman scattering and photoluminescence, can
provide valuable means for tracking, detecting and imaging diseases. They can also help monitor
in vivo therapy status, pharmacodynamical behavior and drug delivery efficiency [26].
1.2.2.1.
I.
Biomedical Applications of Carbon Nanotubes
Carbon Nanotubes as Biosensors
Owing to their exceptional structural, mechanical, electronic and optical properties, CNTs have
been regarded as a new generation nanoprobes (Tîlmaciu and Morris, 2015). Their high aspect
ratio, high conductivity, high chemical stability and sensitivity (Zhao et al., 2002) and fast
electron-transfer rate (Lin et al., 2004) make them exceedingly fit for biosensing applications.
The basic element of CNT-based biosensors is the immobilization of biomolecules on its surface,
therefore enhancing recognition and the signal transduction process.
On the basis of their target recognition and transduction mechanisms, these biosensors are
largely categorized into electrochemical and electronic CNT-based biosensors and optical
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
biosensors. CNTs have been renowned as promising materials for improving electron transfer,
which makes them appropriate for combining electrochemical and electronic biosensors
II.
Carbon Nanotubes for Drug Delivery
Among the different carbon allotropes, CNTs have attracted escalating attention as a highly
competent vehicle for transporting various drug molecules into the living cells because their
natural morphology facilitates non-invasive penetration across the biological membranes (Chen
et al., 2008; Das et al., 2013; Liu et al., 2013; Panczyk et al., 2016).
Generally, drug molecules are attached to CNT sidewalls via covalent or non-covalent bonding
between the drug molecules and functionalized CNT. But each of these processes has advantages
or disadvantages. The covalent interaction makes the drug-loaded CNT stable in both the extraand intracellular compartments, meaning that such a phenomenon has a lack of sustained release
of the drug inside the cellular microenvironment of cancer cells, which is a shortcoming in the
drug delivery system [27].
1.2.3. Graphene and its derivatives
Graphene is a single or few-layered two-dimensional sp2 bonded carbon sheet, which is another
class of sp2 nanocarbon materials and exhibits many outstanding properties in physics and
chemistry.
Since its discovery in 2004, graphene has been extensively studied in many different fields.
Utilizing the interesting optical, electrical, and chemical properties of graphene, various
graphene-based biosensors have been fabricated to detect biomolecules with high sensitivities.
Graphene has a poly-aromatic surface structure with an ultrahigh surface area, which is available
for the efficient loading of aromatic drug molecules via p–p stacking for applications in drug
delivery. Its thermal conductivity and mechanical stiffness are as high as 3000W m-1 K-1 and
1060 GPa, respectively. Recent studies have shown that individual graphene sheets have
extraordinary electronic transport properties. One possible route to harnessing these properties
for biomedical applications is incorporating graphene sheets in a nanocomposite material.
Although pristine graphene has excellent electrical conductivity, it has poor aqueous solubility;
15
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
thus, various derivatives such as graphene oxide (GO), reduced graphene oxide (rGO), few-layer
graphene oxide (FLGO) and chemically changed graphene (CCG) have been developed.
GO and rGO are much more suitable for sol–gel chemistry and effective candidates for the
synthesis of biocompatible nanocomposites. GO sheets, i.e., the oxygenated counterparts of oneatom thick graphene sheets, can be produced as a high surface single layer by the Hummers
method. GO has been applied in several biotechnologies such as biosensors, cellular imaging,
nanoprobes, drug delivery, and others. The functional groups present on the surface and at the
edges of GO collectively act to inhibit electron transfer. This is why GO has low electrical
conductivity; whereas, its reduced form (rGO) exhibits higher electrical conductivity.
Recently, GO-based nanocarriers have gained significant attention for anticancer drug delivery
and imaging due to their high drug loading and effective delivery capacity. Their specific surface
area reaches approximately 2600 m2 g1, which is more than double that of most nanomaterials.
Moreover, unlike pristine graphene, GO exhibits high water dispersibility and endows pHdependent negative surface charge to maintain high colloidal stability.
However, GO can be aggregated in salt media such as protein-rich cell culture media and
phosphate buffered saline. Another interesting property of GO is physisorption via p–p stacking,
which is effective for loading many aromatic drug molecules such as doxorubicin, a potent
anticancer drug. Thus, owing to its small size, intrinsic optical properties, large specific surface
area, low cost, and useful non-covalent interactions, GO is a promising material for biomedical
applications. Furthermore, GO has the ability to release drug molecules upon stimuli such as NIR
light, potentiating its use as a delivery carrier. However, to date, the in vivo behaviors of GO,
such as its blood circulation, inflammation responses, and clearance mechanism, are not fully
understood, which require future intensive studies [28].
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Figure 7. The chemical structure of grapheme and its derivatives
1.2.3.1.
I.
Biomedical Applications of Graphene Oxide
Graphene Oxide as Biosensor
Graphene oxide is capable of dynamically interacting with the probe and/or for the transduction
of a specific response toward the target molecules. This transduction process is achieved by
fluorescence, Raman scattering and electrochemical reaction. On the basis of this, GO are
broadly used as biosensors (Kim et al., 2017; Suvarnaphaet and Pechprasarn, 2017), and here the
most recent works on the progress of GO based nano-architecture in biosensing applications are
discussed: Graphene nanomaterials have been extensively used for the selective electrochemical
sensing of single-and double-stranded DNA (Liu et al., 2012; Tang et al., 2015). The high
sensitivity could be attributed to the excellent electrochemical properties of graphene, the strong
17
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
ionic interaction between the negatively charged (– COOH) groups and the positively charged
nucleobases, and the robust π–π stacking between the nucleobases and honeycomb carbon
framework.
The Rahigi group developed reduced graphene nanowire (RGNW) biosensors for
electrochemical detection of the four bases of DNA (guanine, tyrosine, adenine and cytosine) by
checking oxidation signals of the discrete nucleotide bases (Akhavan et al., 2012).
II.
Graphene Oxide for Drug Delivery
Utilizing the extremely large surface area and available π electrons, graphene is suitable as a
drug carrier. Wang et al. (2012) loaded a high amount of doxorubicin (DOX) on phospholipid
monolayer coated graphene and subsequently observed the sustained release of DOX to a greater
extent, DOX could be loaded on a graphene sheet via physisorption followed by surface
modification by PEG-NH2 in order to enhance stability and compatibility in a biological medium
[29].
1.2.4. Carbon Quantum Dots
Carbon quantum dots (CQDs) are small carbon nanoparticles with sizes less than 10 nm. The
first report on quantum-sized bright and colorful photoluminescence CQDs, published in 2007
by Sun et al., used laser ablation of a carbon target and a surface passivation method. Recently,
CQDs have been extensively studied to gain high fluorescence quantum yield (QY) with facile
synthesis methods.
CQDs have generally been synthesized from organic materials, including natural polymers (e.g.,
chitosan, gelatin, and other sources. Amino acids, apple juice, grape peel, and vegetables have
also been used to produce CQDs. Furthermore, various simple and low cost-effective methods
have been developed for the synthesis of CQD, including laser ablation, electrochemical
oxidation, combustion/thermal microwave heating, supported synthesis, chemical oxidation,
hydrothermal carbonization, and pyrolysis. Furthermore, uniform nitrogen-doped CQDs were
synthesized via a one-step solvothermal process using nitrogen rich solvents, such as N-methyl2-pyrrolidone (NMP) and dimethyl-imidazolidinone (DMEU). A facile chemical method was
18
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
recently developed to synthesize –C(O)OH-modified CQDs, which is considered an innovative
route for acid attack on CNTs. Moreover, the introduction of surface defects, tailoring their size
and chemical modifications have been used to tune the fluorescence properties of the CQDs [30].
Figure 8. Properties and pros of CQDs
1.2.4.1.
I.
Biomedical Applications of Carbon quantum dots (e.g., GQDs)
Graphene Quantum Dots (GQDs) as Biosensors
Recently, GQD-based biosensors have largely been developed for practical applications in
clinical analysis and disease diagnosis. On the basis of excellent photoluminescence (PL), electro
chemiluminescence (ECL) and electrochemical behaviors of GQD, these have been widely used
for detecting bio-macromolecules including DNA, RNA, proteins or glucose molecules with
better selectivity and sensitivity (Xie et al., 2016; Kumawat et al., 2017). Qian et al. (2014)
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
developed DNA probe-functionalized reduced GQDs to detect DNA based on the Furrier
Resonance Energy Transfer (FRET) fluorescence sensing method.
II.
Graphene Quantum Dots (GQDs) for Drug Delivery
Graphene quantum dots possess some unique features, such as a single atomic layer with small
lateral size and an oxygen-rich surface that renders it suitable for loading drug molecules and
enhancing stability in physiological media. In addition, the fluorescent property of GQD makes it
an appropriate platform for the traceable delivery of the drug into the cancer cells (Cheng et al.,
2015; Pistone et al., 2016; Srivastava et al., 2016). Hence, GQDs have been widely used for drug
delivery in various diseases from last decade [31].
1.2.5. Nanodiamonds
NDs are nanocrystals that consist of tetrahedrally bonded carbon atoms in the form of a threedimensional (3D) cubic lattice; thus, this structure imparts the properties of a diamond and an
onion-shaped carbon shell containing a coating of functional groups on its surface (Figure 8).
The sp2/sp3 bonds in NDs are quite flexible, endowing them with the ability to assume two
geometrical forms, i.e., the stretched face of diamond can behave as a graphene plane and the
puckered graphene may become a diamond surface. The intrinsic properties of NDs are of great
interest, and the smaller the size of NDs, the superior their properties. For NDs with a size
smaller than 2 nm, theoretical works predict quantum confinement effects due to an increase in
their band gap. The size and properties of NDs depend on their synthetic method.
20
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Figure 9. Subtypes of nanodiamonds schematic (A) and photographic (B) comparison
NDs can be easily functionalized with different ligand molecules, which are used as platforms
for the conjugation of various biological molecules, chemical compounds and drugs. Due to their
high surface area and ease of functionalization and doping, NDs have been studied as theranostic
agents. The optical properties of NDs are due to the presence of nitrogen-vacancy (NV) defect
centers, a nitrogen atom next to a vacancy, which allow their use as photoluminescent probes.
NV centers are created by irradiating NDs with high energy particles such as electron, proton,
and helium ions, followed by vacuum annealing at 600–800 °C, which both form vacancies that
migrate and get trapped by the nitrogen atoms present in the diamonds. Furthermore, the NV
centers emit bright fluorescence at 550–800 nm. This excellent emission property together with
their low cytotoxicity make NDs a promising fluorescent probe for single-particle tracking in
heterogeneous environments. When functionalized, their biocompatibility is known to be
superior to single-walled and multi-walled CNTs and carbon black [32].
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
1.2.5.1.
I.
Biomedical Applications of Nanodiamonds
NDs as an antibacterial or antimicrobial agents
Hinder/terminate the growth and reproduction of bacteria. NDs have been found to kill grampositive and gram-negative bacteria. Wehling et al. showed that NDs can be an efficient
antibacterial agent based on their surface composition. Their experiment proposed that the NDs
possessing partially oxidized and negatively charged surfaces would have antibacterial property.
As acid anhydride group on surface. Moreover, surface functionalization of NDs with protein
molecules enhances the bactericidal property of NDs. In addition to the above-mentioned
research, another group investigated the antibacterial activity of ultrafine nanodiamond against
gram negative bacteria, i.e., E. coli.
Functionalization of NDs surface was done with carboxyl group to form carboxylated
nanodiamond (cND) and was kept in highly nutritious media. Upon scanning electron
microscopy (SEM), the photomicrograph revealed that cND was attached to the bacterial cell
wall surface leading to its destruction. Surface functionalization of NDs with glycan (sugar
coating) had also uncovered the bactericidal effect of NDs specifically for type 1 fimbriaemediated E. coli. adhesion. These have the potential in countering E. coli. biofilm formation.
NDs form covalent bond with molecules on cell walls or bind to intracellular components which
inhibit vital enzymes and proteins, leading to a rapid collapse of the bacterial metabolism and
finally cell death.
II.
NDs in Gene Therapy
Gene therapy can be used in treatment of various life-threatening diseases, like cancer, heart
disease and diabetes. NDs act as an emerging attractive tool for gene delivery, by which
efficiency of gene therapy is much more increased. The technology requires both effective
cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an
example, Chu et al. demonstrated the successful cytosolic delivery and expression of such a gene
using the prickly NDs as carrier. Perevedentseva et al. provided evidence that lysine
functionalization enables NDs to interact effectively with the biological system to be used for
RNAi therapeutics. Zhang et al. demonstrated NDs as viral vectors for in vitro gene delivery via
22
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
surface immobilization with 800 Da polyethyleneimine and covalent conjugation in presence of
amine groups. This approach represented an efficient avenue towards gene delivery via DNA
functionalized NDs.
NDs have also been explored their potential in the delivery of small interfering RNAs. Liu et al.
investigated the potential of small interfering RNAs (siRNA) loaded functionalized NDs with
polymer polyethylenimine (PEI) for its in vitro efficiency and cytotoxicity via simulation
technique. The results showed to be highly effective for in vitro delivery with low cytotoxicity.
In addition, Alhaddad et al. elucidated the delivery of siRNA via cationic polymers viz.
polyallylamine and polyethylenimine coated diamond nanocrystals. They targeted Ewing
sarcoma cells which were traceable for long time owing to their intrinsic fluorescence.
III.
Carrier for Drug and Peptide Delivery
For the conjugation of active pharmaceutical ingredients, NDs are ideal candidate owing to their
huge surface area and surface functionalities. A drug carrier is found to be suitable only in terms
of its loading capacity, capability of protection from surrounding environment and inert nature.
A prominent drug loading efficiency with less concentration of carrier is highly appreciated.
Simultaneously, timely release of drug from the carrier is also of great significance for desired
therapeutic effect. Huang et al. investigated the loading and release of a chemotherapeutic agent
viz. doxorubicin hydrochloride (DOX) from NDs. The research was based on the concept of
ionic interaction between carboxylic and hydroxylic groups present on the surface of NDs and
amine group of DOX to form NDs-DOX loose cluster. In further studies, it was found that DOX
was adsorbed on surface of NDs and also in the fissures of cluster. Additionally, cytotoxic
studies of DOX-NDs on mouse macrophages and human colorectal cancer cells revealed a lower
toxic effect with sustained release than free-DOX.
Apart from the large surface area for conjugation, NDs have also emerged as dispersibility
enhancing agents of hydrophobic drugs. There are certain chemotherapeutic moieties which have
their solubilities in organic solvents that limit their parenteral administration viz. a liver cancer
drug ‘purvalanol A’ and a breast cancer moiety ‘4-hydroxytamoxifen’. The characteristic of
enhancing dispersibility in water is attributed to NDs' nature of adsorbing drug on surfaces and
retaining therapeutic effectiveness of the drug. The therapeutic activity of NDs formulations was
23
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
confirmed by MTT assay. These outcomes revealed that NDs could play a significant role in
formulation development of poor water-soluble drugs [33].
1.3.
Toxicity of Carbon-based Nanomaterials
Carbon nanomaterials are a novel class of materials that are widely used in biomedical fields
including the delivery of therapeutics, biomedical imaging, biosensors, tissue engineering and
cancer therapy. However, they still suffer from their toxic effect on biological systems. Until
now, various investigations have been carried out on the toxicity of CNT (Liu et al., 2013;
Madani et al., 2013; Allegri et al., 2016; Kobayashi et al., 2017).
From numerous studies it has been revealed that several factors contribute to the toxicity of
CNT. The effect of metal impurities in CNT could have a substantial impact on toxicity
(Koyama et al., 2009; Vittorio et al., 2009; Aldieri et al., 2013). The impurities, such as metal
ions, were incorporated inside the CNT during synthesis and caused toxicity to the cells. The
length of CNT has a great impact on the toxicity of CNT only due to the failure of their cellular
internalization (Kostarelos, 2008). Some groups have prepared CNT with different sizes and
studied their toxic behavior on cells or DNA (Smart et al., 2006; Raffa et al., 2008). The
Donaldson group described that long-term retention of long CNT led to severe inflammation,
which caused progressive fibrosis (Murphy et al., 2011). Moreover, the higher diameter with
equal average length of CNT exhibits greater toxicity (Kolosnjaj-Tabi et al., 2010).
Figure 10. The mechanism by which carbon based nanomaterial induce cytotoxicity
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Owing to the difference in size, structure and chemical surface states between SWCNT and
MWCNT, they delivered different toxicity effects on cells (Fraczek et al., 2008; DiGiorgio et al.,
2011). Moreover, the solubilizing agents played an important role in the toxicity of CNT (Nam et
al., 2011; Kim et al., 2012). The individual CNTs tend to bundle in presence of some natural
dispersants and led to toxicity. Interestingly, surface functionalization of CNT triggered toxicity
in cells. The Jos group found that (– COOH) functionalized SWCNT induced higher toxicity
compared to the non-functionalized SWCNT in the HUVEC cell lines (Praena et al., 2011). On
the other hand, Li et al. (2013) demonstrated that strongly cationic functionalized MWCNT has
greater potential for lysosomal damaging due to their high cellular uptake and NLRP3
inflammasome activation in comparison to the carboxyl group-functionalized or moderately
amine group-functionalized MWCNT, as can be observed by confocal imaging (Figure 5A; Li et
al., 2013).
Like CNT, graphene has also limitations to biomedical application due to its toxicity. Ou et al.
(2016) thoroughly described in their recent review article the toxicity of graphene in different
organs. Numerous studies have been conducted on the toxicity of graphene in animals and cells
(Shareena et al., 2018). It was stated that several parameters, including concentration, lateral
dimension, surface property and functional groups, greatly influence its toxicity in biological
systems (Seabra et al., 2014; Alshehri et al., 2016). Li et al. (2014) observed that GO at a
concentration of 100 mg/L induced reactive oxygen species (ROS) production in GLC-82 cells
upon incubation for 24 h and caused toxicity. To overcome the toxic effect of GO in various
biomedical applications, many research groups have designed GO with various biological
molecules. The Zhou group modified a graphene sheet by coating it with blood protein to reduce
its toxic effect (Chong et al., 2015). Among different materials of the carbon family, GQDs
contain some exciting properties and these have thus been extensively used for biological
applications as discussed above.
The toxicity of GQDs is different from graphene and GO, thus it is an imperative and serious
issue that ought to be addressed. After many investigations, it has been implied that various
parameters govern the toxicity of GQDs. It seems that the smaller size of GQDs is an advantage
25
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
over GO or CNT in terms of toxicity. More importantly, Wang et al. (2016) showed a cell
viability mapping curve for various cells under the same conditions and concluded that GQDs
with a size below 10 nm possess extremely high cell viability. No doubt, the concentration of
nanomaterials is a dominating factor in toxicity. For GQDs, the concentration tolerance of the
cells to different GQDs is contradictory. The Shen group showed theoretically that the potential
cytotoxicity of GQDs depends on their size and concentration (Liang et al., 2016). They
observed that in the 100 ns scale simulation, GQDs with relatively small size could permeate into
the POPC membrane. The permeation of GQDs could affect the thickness of the POPC lipid
membrane. At the starting point, angles between GQDs and lipid membrane were 0° in all cases.
During simulation, smaller-size GQDs permeated the POPC membrane and created an angle in
the range between 45° and 70°. GQDs with larger sizes were only absorbed on the lipid
membrane surface and formed an angle in the range of 0° to 10°.
Moreover, it has been observed that the surface functional groups of nanomaterials have a great
impact on the toxicity of nanomaterials. The Shang group reported after an investigation that
hydroxylated-GQDs have significant toxicity on A549 and H1299 cells (Tian et al., 2016). In
contrast, Nurunnabi et al. (2013) claimed that carboxylated GQDs had no acute toxicity on
different cancer cells such as KB, MDA-MB231, A549 and the normal cell line such as MDCK.
Furthermore, after a long-term in vivo study they did not find notable damage to the organs.
Regrettably, we have not yet found any article that gives clear information based on the effect of
different functional groups in the toxicity of GQD nanomaterials [34].
26
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Figure 11. Scheme of possible toxic effects of nanoparticles. The main mechanism of
nanoparticle toxicity is via oxidative stress and increase in ROS levels.
Figure 12. Toxicity mechanism of nanoparticles mediated by reactive oxygen species (ROS)
generation. The model describes extracellular sources of ROS as exposure routes for the
engineered nanoparticles. Intracellular ROS can be generated from the mitochondria, which
later causes lipid peroxidation, DNA damage and protein denaturation.
27
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Chapter 2
2.
2.1.
CNTS in neuroregeneration and neurodrug delivery
Carbon nanotubes as nerve tissue reconstructing platforms
Carbon nanotubes are cylindrical nanostructures made up of graphene sheets wrapped onto
themselves . In neuroscience applications, the mostly used geometries are single-walled carbon
nanotubes (SWCNT), made up of a single graphene sheet rolled-up and closed at its ends by
hemispheric fullerene caps, and multi-walled carbon nanotubes (MWCNT), made up of several
concentric graphene cylinders. Currently, carbon nanotube-based applications in neuroscience
include: electrical interfaces for neuronal stimulation and recording (that drastically improve the
electrode performance, both in vitro and in vivo as well as platforms to promote neuronal
survival, differentiation, growth and performance.
Starting more than 10 years ago, numerous studies reported the impact of carbon nanotubes on
neuronal behaviour and, in particular, on their ability to promote both neurite extension and the
development on neuronal electrical features, at both the single-cell and neuronal network levels.
28
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
In this respect, carbon nanotubes have been successfully applied in in vitro studies following two
different strategies, namely using them as scaffolds (substrates) for neuronal growth or as
soluble factors . In both cases, carbon nanotubes applications had an unexpected and exciting
impact on neuronal signalling and behaviour.
2.2.
Carbon nanotubes ability in guiding neuronal cells' behaviour
2.2.1. Carbon nanotube-based devices to promote growth and neurite elongation
The first in vitro study reporting the biocompatibility of carbon nanotube for neuronal growth
was that of Mattson and colleagues , who demonstrated the ability of MWCNT-layered
substrates (in the form of films of tangled MWCNTs) to support the long-term survival of
cultured dissociated hippocampal neurons. Noteworthy, this work was the first that highlighted
the importance of carbon nanotube functionalization in modulating neuronal behaviour. In
particular, by non-covalently modifying MWCNTs with 4 hydroxynonenal, it was possible to
discern their effect from that of non-functionalized (pure) MWCNTs, demonstrating that 4hydroxynonenal-functionalized MWCNTs were more effective in inducing the construction of
elaborated neuritic arborisation (displaying increased length and higher branching). Conversely,
neurite growth on MWCNT was reduced when compared to polyethyleneimine (PEI)-coated
substrates, used as controls . A later study directly compared growth substrates of purified
MWCNT tangled films with a (glass) control substrate, demonstrating that such nanomaterial
meshworks are as good as controls in supporting neuronal survival in vitro, as the density of
dissociated hippocampal neurons cultured in the two conditions was the same. The same work
showed that, compared to the control substrate, pure MWCNTs did not promote the extension of
new neurites, as the number of neurites per cell was the same in the two conditions . More
recently, purified carbon nanotube scaffolds have been shown to be biocompatible also when
interfaced to more complex, three-dimensional neuronal systems (in vitro cultured spinal
explants ). In this model, purified MWCNT films are able to significantly promote the outgrowth
of sensory or motor axons (emerging from dorsal root ganglia – DRG – neurons and/or
motoneurons), as explants interfaced for two weeks to the artificial scaffolds showed both an
29
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
increased number and length of SMI-32 positive neuronal fibres. Interestingly, MWCNT
scaffolds could also modulate the elastomechanical properties of neuronal fibres . The promotion
of neurite elongation by MWCNTs in this in vitro system raises the issue of possible differences
in carbon nanotubes effects on neurite outgrowth when interfaced to central or peripheral axons.
This issue has not been systematically investigated yet and requires future studies.
The biocompatibility of unmodified carbon nanotube in in vitro systems has been confirmed by
several authors, in studies where carbon nanotubes were employed as substrates for neuronal
growth in the form of tangled nanotube films or islands or as vertically aligned fibres .
Regardless of the use of pure nanotubes in in vitro studies, functionalizing carbon nanotubes
with various bioactive moieties is a prerequisite for future applications in vivo, to favour carbon
nanotube biocompatibility in terms of degradation and elimination from the body. Besides,
functionalizing carbon nanotube provides a powerful tool to design new carbon nanotube-based
scaffolds with improved performance in promoting neuronal growth and neurite elongation. In
agreement with the known ability of positively-charged substrates, e.g. polylysine or
polyornithine, in promoting neuronal attachment,neuronal survival and neurite extension, a work
published in 2004 demonstrated that tuning MWCNTs electrical charges strongly affected
cultured neurite outgrowth. In this study, Hu and colleagues functionalized MWCNTs with
negatively charged (\COOH), neutral (poly-m-aminobenzene sulfonic acid: PABS, zwitterionic)
and positively charged (ethylenediamine: EN) moieties and tested these modified MWCNTs as
growth support for dissociated hippocampal neurons.
While the differently charged tubes had no impact on the number of neurites emerging from the
soma, the average neurite length was strikingly higher (almost doubled) in neurons grown on the
positively charged MWCNT-EN. Furthermore, neurite branching progressively increased from
negatively charged (MWCNT-COOH), to neutral (MWCNT-PABS), to positively charged
(MWCNT-EN) substrates (Fig.13).
30
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Figure 13. MWCNT electrical charge modulates neurite outgrowth. Schematic
drawing summarizing the effects of different MWCNT scaffold electrical charges
on outgrowing neurite length and branching.
This study demonstrated, for the first time, that it is possible to control neurite extension and
branching by chemically modifying carbon nanotubes , and paved the way for exploiting this
strategy to optimize carbon nanotube sophistication. In addition to surface charge, electrical
conductivity is an additional property relevant in manufacturing carbon nanotube based
scaffolds: neurite outgrowth is boosted only by carbon nanotubes within a narrow range of
conductivity. Malarkey and colleagues sprayed a polyethylene glycol (PEG) functionalized
carbon nanotube solution onto glass supports to produce films of different thicknesses that
differed in terms of conductivity (0.3, 28 and 42 S/cm), with equal average roughness. These
films were used as substrates for the growth of dissociated hippocampal neurons. While the
variation in conductivities did not affect the number of neurites emerging from the soma or the
number of growth cones, the neurite length in neurons grown on the scaffold with the smallest
conductivity (0.3 S/cm) was strikingly higher (almost doubled) than in neurons grown on PEGcarbon nanotubes with larger conductivities or on controls substrates (polyethyleneimine). The
conductivity and surface charge of carbon nanotubes are therefore extremely important for the
impact of such scaffolds on neuronal outgrowth, and might therefore be finely tuned to maximize
the carbon nanotube ability to impact on neuronal growth. Noteworthy, this finding suggests that
differences in conductivity may explain, in this respect, the variable outcomes reported by
different studies.
31
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
The extracellular environment plays a critical role in the modulation of cellular physiology and
growth, and ongoing research wishes to design new strategies to take advantage of the ECM
components toward promoting neuro-regenerative environment.
In fact, recent studies explored the possibility of manufacturing scaffolds based on carbon
nanotubes doped with ECM molecules, with the aim of enhancing the effects of natural and
artificial cues on neuronal behaviour.
SWCNT/type I collagen and MWCNT/type IV collagen blends are biocompatible matrices for
the in vitro growth of PC 12 cells, but did not show any significant impact on neurite extension.
Nevertheless, the combination of the MWCNT/type IV collagen matrix to the electrical
stimulation of PC12 cells (via the conductive matrix itself) was able to induce PC12 cell
differentiation into neurons and, therefore, promote their neurite extension.
2.2.2. Patterned carbon nanotube platforms to spatially direct neuronal growth
In addition to neurite outgrowth-promoting features, effective neuroregenerative strategies would
benefit from the possibility to specifically, spatially direct neurite growth. In addition to the
biochemical composition of the extracellular environment, neuronal growth, neuritogenesis and
neuronal polarity are extremely sensitive to the substrate topography, at both the micro- and the
nanoscale.
Accordingly, carbon nanotube-based growth substrates combined to micro patterning techniques
have been successfully exploited to spatially direct neurite growth.
In 2005, Zhang and colleagues combined microlithography and chemical vapour deposition
techniques to fabricate substrates made of vertical MWCNT arrays arranged in geometricallypatterned substrates.After poly L-lysine coating, the scaffolds were used as growth substrates for
guiding neurite growth in a hippocampal neuronal cell line. Neurite growth followed the edges of
the patterned substrate ,thus providing the first evidence of the ability of carbon nanotubes to
direct neurite elongation . This ability of patterned carbon nanotube substrates was confirmed by
later studies . Interestingly, in the first phases of in vitro growth, major neurites closely following
the nanotube patterns grow faster than those extending in random directions; surprisingly,
32
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
neurites followed the nanotube patterns only until a certain length then they deviated in other
directions.
Highly oriented carbon nanotube sheets or yarns have also been employed as biocompatible
substrate for in vitro growth of a variety of cell phenotypes, including cortical, cerebellar and
dorsal root ganglia neurons, the latter showing processes closely following the surface topology
of nanotube yarns . Neuronal processes from cortical and cerebellar neurons grown on oriented
carbon nanotubes display comparable length than those grown on polyornithine; however, the
tips of the growing neurites have growth cones with an almost doubled area in carbon nanotube
substrates, thus suggesting an increased activity of these guidance organs in sensing
nanotopographical cues. This finding may suggest the use of carbon nanotubes as effective tools
to boost neurite sensitivity to the growing substrate.
Recently, carbon nanotubes have also been combined to other synthetic nano-materials in the
design of scaffolds which are able to favour a certain directional neurite growth. In the works of
Jin and colleagues, carbon nanotubes have been successfully employed as a coating nanomaterial
that strongly improves the ability of PC12 and DRG neurites to grow along poly (L-lactic acidco-caprolactone) nanofibres.
Although these results have been obtained in simplified biological models (in vitro dissociated
neurons) and are far from neuronal growth in natural, complex tissues, they support carbon
nanotube-based scaffolds as suitable substrates for the development of devices enriched with an
efficient and spatially-directed neurite re-growth.
2.2.3. Carbon nanotube growth substrates ability to improve nerve cell performance
Neuroregeneration and recovery of lost functions require axons (re)growth on the one hand and
the establishment of functional synapses or the improvement of neuronal signal transferring
ability, on the other.
One of the carbon nanotube's distinct properties is their high electrical conductivity that,
regardless of morphological changes and via direct interactions with neuronal membranes, may
33
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
impact on neuronal electrogenic properties. Recent in vitro studies showed that growth scaffolds
of pure SWCNTs and MWCNTs were instructive for neuronal electrical behaviour. More
explicitly, carbon nanotubes were able to improve neuronal electrogenic properties in developing
neurons at three different levels of complexity: single-cell , synaptic and three dimensional tissue
levels. The analysis of singe-neuron electrophysiological properties showed that carbon nanotube
scaffolds that were used as substrates for the growth of dissociated hippocampal neurons do not
change neuronal passive properties (capacitance, input resistance and resting membrane
potential, indicators of cellular health and dimensions), but they strongly impact on single-cell
regenerative electrical properties, i.e. on neuronal integrative ability . Neurons interfaced to
carbon nanotubes, when forced to fire action potentials at a relatively high frequency, are more
prone to generate backpropagating action potentials, a neuronal regenerative property involved in
local synaptic feedback regulation and messengers release , finally boosting their single-cell
excitability . Strikingly, the observed phenomenon is dependent on both nanotube conductivity
and nanotopography, as growth substrates presenting only either conductivity or nanotopography
comparable to the carbon nanotubes (indium tin oxide or RADA peptide, respectively) were
unable to improve action potential backpropagation . The mechanism proposed to explain these
observations, supported by theoretical modelling and by the evidence of numerous tight and
intimate contacts between carbon nanotubes and neuronal membranes, is the presence of an
“electrical shortcut” between adjacent dendritic compartments mediated by the electrically
conductive substrate . The presence of an electrical coupling between nanotubes and cell
membranes is indeed sustained by other studies ; however, the mechanism mediating the
observed effect has not been demonstrated yet.
The effects of MWCNT scaffolds on single-neuron excitability are paralleled by a strong impact
on synaptic network activity. On dissociated hippocampal neurons, which spontaneously
reconstruct synaptically active neuronal networks in vitro, carbon nanotube scaffolds are able to
increase the frequency of spontaneous synaptic activity, measured as post-synaptic currents,
while simultaneously increasing the spontaneous action potential firing frequency (Fig. 14) .
34
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Figure 14. Carbon nanotube scaffolds increase spontaneous synaptic activity.
Representative traces obtained by patch-clamping dissociated hippocampal
neurons cultured on control glass or on a MWCNT scaffold, showing increased
frequency of both spontaneous post-synaptic currents (A) and action potential
firing frequency (B) in neurons grown on MWCNT.
Importantly, this effect is independent from any change in neuronal density or morphology, as
the number of neurons adhering to the conductive scaffold, their somatic size and neurite number
are comparable to those of controls. An explanation of the observed phenomena was proposed by
another recent study, reporting the ability of carbon nanotube scaffolds to boost the formation of
functional synapses. In cultured hippocampal networks the number of synaptic connections
(measured via quantifying synaptically coupled neuron pairs recorded electrophysiologically and
via synaptic contact reconstruction by immunofluorescence) is markedly increased when neurons
are interfaced to MWCNTs with respect to controls . Furthermore, MWCNTs are also able to
affect the short-term dynamics of neuronal synaptic transmission: when activated repetitively,
synapses developed on nanotubes did not undergo short term synaptic depression, thus
strengthening their ability to transfer information.
35
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
A step beyond the study of carbon nanotube technology applications came in 2012, by
employing MWCNT scaffolds as growth substrates for three-dimensional spinal explants . In this
model, MWCNT growth platforms modulated the functional performance of neurons spatially
far from the scaffold: the study demonstrated, at the level of cells not directly in contact with the
interface, an increase in the spontaneous synaptic activity (measured as post-synaptic currents
amplitude) and in the evoked afferent responses . It seems that physical properties expressed by
carbon nanotube substrates can really play an active role in the design of neuro-prosthetic
devices improving axonal regeneration and electrical neuronal responses, and transferring their
effects to regions of the tissue relatively far from the interface itself. Another important feature
of carbon nanotube based growth platforms is their ability to accelerate the onset of neuronal
electrical activity.
By culturing rat hippocampal neurons on multi-electrode arrays (MEAs) in which SWCNTs
were deposited at the microelectrodes tips, Khraiche and collaborators showed the appearance of
electrical network activity as soon as 4 days of culturing, while no activity was present in control
cultures (grown on bare gold electrodes) till day 7, suggesting a SWCNT-induced enhancement
of neuronal excitability . A recent study strengthened the idea that nanotube scaffolds foster the
development of mature neuronal phenotype, by focussing on a critical step in neuronal
maturation of the fast inhibitory transmission system, i.e. the chloride shift. During development,
neuronal expression of the potassium chloride cotransporter 2 (KCC2) increases, thus boosting
chloride extrusion and converting the action of GABA from excitatory to inhibitory. Dissociated
embryonic cortical neurons were cultured on a few-walled carbon nanotube (fwCNT)–arabic
gum matrix coated with polyornithine (or on a standard polyornithine control substrate), and
tested for their KCC2 expression and chloride-extrusion ability. Neurons growing on the
fwCNT–arabic gum matrix showed an accelerated chloride shift as a consequence of an
increased expression of the KCC2 cotransporter, a finding confirmed also in brain slice cultures
interfaced to the fwCNT–arabic gum matrix. Importantly, also in this case this phenomenon is
fwCNT-specific, in fact the substitution of fwCNT with a nanomaterial of similar structure, but
lacking electrical conductivity (SiOx) is unable to replicate the maturation-accelerating effect of
the fwCNT-based matrix.
2.3.
Mechanisms underlying carbon nanotube-mediated neuromodulation
36
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Increasing evidences point to a combination of the different proposed mechanisms. In general,
neuritogenesis, neurite growth and neuronal polarity are strictly governed by the complex
intertwining of architecture of ECM components and their spatial gradients , and by the
topographical features of the neuronal growing environment that underlines neuronal membrane
mechanics and also triggers specific intracellular signalling cascades . Accordingly, a particular
role in the neuronal interaction with the carbon nanotube environment has been demonstrated in
the case of focal adhesions, the integrin-mediated adhesion structures which allow cells to scan,
sense and react to the surrounding physical environment, and which are finely tuned in their
formation and maturation by substrate nanotopographical features . An increase in the focal
adhesion kinase (FAK) expression was shown in PC12 cells cultured on MWCNT-coated PLCL
(poly(L-lactic acid-co-caprolactone) nanofibres . The extensive adhesion processes between
neuronal membranes and carbon nanotubes are mirrored by the characteristic morphological
adaptations of membranes to these scaffolds, involving high levels of neuronal processes curling
and entanglement around carbon nanotube nanostructures . It has been suggested that carbon
nanotube dimensions and roughness closely matching the diameter of neuronal processes are
fundamental features allowing neuronal membrane binding to the surface.
The “biochemical hypothesis” is supported by the findings that layers of pure, nonfunctionalized and non-coated carbon nanotubes (as single/double-walled carbon nanotubes
films or as MWCNT compacts) show a marked ability to adsorb proteins . The hypothesis is that
carbon nanotube scaffolds act as a porous network of elements forming a rich reservoir of
proteins and growth factors, an ability which is positively correlated to the film thickness (with a
saturation around 70 nm thickness) and relies on the bulk carbon nanotube network, rather than
on surface roughness only.
Nanotopography (mediating neuronal adhesive processes) and/or protein adsorption ability are
not the only factors that make carbon nanotubes so effective in modulating neuronal behaviour:
several recent studies pointed to their electrical conductivity as a critical player in mediating their
impact on both neuronal growth and electrical behaviour. In fact, it has already been reported
that carbon nanotube conductivity exerts a critical role in neuronal growth and in boosting
neuron electrical performance, shown by the fact that a non-conducting nanomaterial-based
scaffold with nanotopography similar to that of the carbon tubes completely lacks growth37
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
promoting or electrical promoting effects on neurons. How carbon nanotube high conductivity
can affect neuronal growth is still not clear; the observed tight contacts between MWCNT or
SWCNT bundles and neuronal membranes favour the hypothesis of a direct electrical coupling.
In this respect, there are no indications for the existence of an optimal conductivity range for
carbon nanotube boosting of neuronal electrical performance, as no systematic study of the
relation between this substrate conductivity and neuronal activity has been published. Not
withstanding the important findings reported above, further research is still needed to fully
explain the fine mechanisms underlying this synthetic material ability to ultimately affect neurite
extension, synapse stabilization and neuronal electrical properties.
2.4.
CNTs in drug delivery
Neuroprotection could be achieved in the future by use of nanodrug delivery in chronic
neurological disorders. Delivery of drugs across the blood–brain barrier is attained by the
application of nanotechnology in therapeutic techniques, known as nanomedicine. Recent
research shows that the nanomedicine required for neurodegenerative pathologies is much less
than that needed for cancer and infectious diseases. Thus, emerging nanotechnologies for
production of neurotrophin delivery systems are promising in terms of their ability to activate
neurotrophin signaling for neuroprotection and neuroregeneration. Neurotrophins are proteins
that were initially recognized as being factors related to the survival of sympathetic and sensory
neurons. Neurotrophins are essential for the development and function of neurons in both the
CNS and PNS, and can be delivered using CNTs.
The use of CNTs as a delivery mechanism for the treatment of CNS pathology is based on their
structural features, especially their improved solubility in physiological solvents (even though
not a heterogeneous solution) due to their functionalization, large surface area, ability to be
easily modified with drug molecules, and biocompatibility with neural systems. Zhang et al used
SWNTs (Single-Walled Nanotubes) modified with acetylcholine to treat AD (Alzheimer’s
Disease). After gastric gavage, SWNT doses up to 300 mg/kg could enable delivery of drug into
the lysosomes of neurons, thus demonstrating the effectiveness of this therapy. Dealing with
brain tumors is a challenge regardless of the therapeutic advances made with the clear
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
understanding of carcinogenesis. Anti-tumor drug molecules have low permeability across the
blood–brain barrier, and this has opened up new possibilities for CNT-based approaches. In one
study, a drug delivery system using CNTs considerably enhanced the effects of CpG
oligodeoxynucleotide immunotherapy in the treatment of glioma (a tumor arising from the glial
cells) and preventing tumors. CNT-based therapy would be useful in the treatment of a number
of neurodegenerative pathologies. SWNT functionalized with amine groups via the amidation
reaction enhances the tolerance of neurons to ischemic injury. Using this method, neurons are
protected and their functions are regained with amine-modified SWNT without therapeutic or
drug molecules. The mechanism via which amine-functionalized SWNTs protect neurons is as
yet unclear. A study by Al-Jamal et al demonstrated the effectiveness of amino-functionalized
MWNTs (Multi-Walled Nanotubes) in delivery of small interfering RNA that decreased
apoptosis at the injury site and promoted recovery in a rodent model of endothelin-1 stroke. The
findings of research on the use of CNTs for neuroregeneration are summarized in Table 1.
Abbreviations: CNT, carbon nanotube; MWNT, multi-walled nanotube; PEG, poly (ethylene
glycol); SWNT, single-walled nanotube.
Table 1. Evidence for application of carbon nanotubes in neuroregeneration
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
2.4.1. Delivery of drugs for various therapies
Carbon nanohorns (CNHs) are distorted horn-like shaped CNTs which are spherical aggregates.
Investigation studies have demonstrated that CNTs and CNHs are a prospective drug delivery
system carrier. As per potential carrier system in biomedical applications, the CNT based on
gelatin (hydrogel) combination has also been used. Nanotubes are used to treat cancer as a drug
delivery carrier. And they are identified for amphotericin B targeting cells. Polyphosphazene
platinum, the anticancer drug administered by means of nanotubes had improved permeation,
bio-distribution as well as retention within the brain because of regulated nanotube lipophilicity.
2.4.2. Diagnostic tools
Nanotubes applications cover different arenas for instance medication, electronics,
industrialization, nanotechnology, etc. Here are certain obstacles including the functionalization,
pharmacology, as well as toxicity of nanotubes that must also be tackled beforehand CNTs are
used in biomedical and biological environments as illustrated in Table 2.
Table 2. Application of CNTs as diagnostic tools
2.4.3. Genetic engineering
Nanotubes and nanohorns have been utilized in genetic engineering to manipulate the genetic
material and atoms in the advancement of proteomics, tissue engineering, genomics, in addition
to bioimaging. Nanotubes are used as genes carrier (gene therapy) in the treatment of various
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
genetic disorders and also cancer because of their cylindrical structure and characteristics as
illustrated in Table 3.
Table 3. Application of CNTs in genetic engineering
2.4.4. Artificial implants
In general, the body demonstrates a pain post-administration along with rejection reaction for
implants. But then, by using other proteins and amino acids, miniature-sized CNHs and CNTs
are anchored to circumvent rejection. They can also be used as implants without host rejection
reactions as an artificial joint. In addition, calcium filled nanotubes which are
arranged/congregated into the bone edifice could also enact as a substitute of bone owing to its
high tensile strength as illustrated in Table 4.
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Table 4. Application of CNTs as artificial implants
2.4.5. As catalyst
Carbon nanohorns proposes a huge surface area and therefore the catalyst next to the molecular
level could be assimilated in bulk quantities of nanotubes and could also be released at a specific
time and at the required rate simultaneously. It is, therefore, possible to reduce the frequency and
quantity of the addition of catalysts by using CNTs and CNHs.
2.5.
Cellular uptake of carbon nanotubes
An important characteristic of f-CNT is their high propensity to cross cell membranes. CNT
labelled with a fluorescent agent were easily internalized and could be tracked into the cytoplasm
or the nucleus of fibroblasts using epifluorescence and confocal microscopy. The mechanism of
uptake of this type of f-CNT appears to be passive and endocytosis-independent. Incubation with
cells in the presence of endocytosis inhibitors did not influence the cell penetration ability of fCNT. Furthermore, f-CNT showed similar behavior when incubation with the cells was carried
out at lower temperatures. Cellular uptake was confirmed by Dai and colleagues [ref] who in
later studies used oxidized CNT to covalently link fluorescein or biotin, allowing for a biotin–
avidin complex formation with fluorescent streptavidin. Again, the nanotubes were observed
inside the cells. In this case, the protein–CNT conjugates were found in endosomes, suggesting
an uptake pathway via endocytosis. The CNT can also be visualized inside the cells using
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
transmission electron microscopy (TEM). Functionalized water-soluble CNT were incubated
with HeLa cells. The cells were subsequently embedded into an epoxy resin that was sliced using
a diamond microtome. Each slice was mounted on a TEM grid and observed under the
microscope. Figure 15 shows a typical example of functionalized MWNT distributed into the
cytoplasm.
Some tubes were also identified at the cell membrane during the process of translocation. The
conformation of CNT perpendicular to the plasma membrane during uptake suggested a
mechanism similar to nanoneedles, which perforate and diffuse through the lipid bilayer of
plasma membrane without inducing cell death. Dynamic simulation studies have shown that
amphiphilic nanotubes can theoretically migrate through artificial lipid bilayers via a similar
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Figure 15. Ultrathin transverse section of HeLa cells treated with functionalized
Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
mechanism [ref]. Nano-penetration was also recently suggested by Cai et al., who proposed an
efficient in vitro delivery technique called nanotube spearing [ref]. MCF-7 breast cancer cells
were grown on a substrate and incubated with magnetic CNT. A rotating magnetic field first
drove the nanotubes to spear the cells. In a subsequent step, a static field pulled the tubes into the
cells. On the basis of SEM images, it seems that the tubes cross the cell membrane like tiny
needles. Another efficient way to observe CNT intracellularly was developed by Weismann et
al., who used near-infrared fluorescence. They showed that macrophage cells could ingest
significant amounts of nanotubes without apparent toxic effects. The internalized tubes remained
fluorescent and could be identified at wavelengths beyond 1100 nm. Therefore, there is
mounting evidence that f-CNT are capable of efficient cellular uptake by a mechanism that has
not yet been clearly identified. However, the nature of the functional group at the CNT surface
seems to play a determinant role in the mechanism of interaction with cells.
The search for new and effective drug delivery systems is a fundamental issue of continuous
interest. A drug delivery system is generally designed to improve the pharmacological and
therapeutic profile of a drug molecules. The ability of f-CNT to penetrate into the cells offers the
potential of using f-CNT as vehicles for the delivery of small drug molecules. However, the use
of f-CNT for the delivery of anticancer, antibacterial or antiviral agents has not yet been fully
ascertained. The development of delivery systems able to carry one or more therapeutic agents
with recognition capacity, optical signals for imaging and/or specific targeting is of fundamental
advantage, for example in the treatment of cancer and different types of infectious diseases. For
this purpose, we have developed a new strategy for the multiple functionalization of CNT with
different types of molecules. A fluorescent probe for tracking the cellular uptake of the material
and an antibiotic moiety as the active molecule were covalently linked to CNT. MWNT were
functionalized with amphotericin B and fluorescein.
The antibiotic linked to the nanotubes was easily internalized into mammalian cells without toxic
effects in comparison with the antibiotic incubated alone. In addition, amphotericin B bound to
CNT preserved its high antifungal activity against a broad range of pathogens, including Candida
albicans, Cryptococcus neoformans and Candida parapsilosis.
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
In an alternative approach by a different group, SWNT have been functionalized with substituted
carborane cages to develop a new delivery system for an efficient boron neutron capture therapy.
These types of water-soluble CNT were aimed at the treatment of cancer cells. Indeed, these
studies showed that some specific tissues contained carborane following intravenous
administration of the CNT conjugate and, more interestingly, that carborane was concentrated
mainly at the tumor site.
In view of these results, f-CNT represent a new, emerging class of delivery systems for the
transport and translocation of drug molecules into different types of mammalian cells. Although
these CNT conjugates displayed no cytotoxicity in vitro, for further development, it will be
important to assess their metabolism, bio-distribution and clearance from the body.
https://www.sciencedirect.com/science/article/pii/S1367593105001389
Figure 16. Schematic illustration of the drug delivery process. (a) CNT surface is
linked with a chemical receptor (Y) and drugs (●) are loaded inside, (b) open end of
CNT is capped, (c) drug-CNT carrier is introduced in the body and reaches the target
cells due to chemical receptor on CNT surface, (d) cell internalizes CNT by cell
receptors (V) via endocytosis pathway for example, € cap is removed or biodegrades
inside the cell, then drugs are released.
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
2.6.
Limitations and Challenges
2.6.1. Lack of solubility/dispersion
The lack of solubility of CNTs has been reported in various literatures and it has posed as a
major limitation to its utilization. Multitude of approaches for enhancing the solubility and
dispersion of CNTs has been developed. Carbon nanotubes’ insolubility is usually caused by
their hydrophobic structure, the distinct surface area, forces such as van der Waals etc. There can
be only one alternative for these problems that researchers have found (insolubility and poor
dispersion), and that would be an alteration of the nanotubes by functionalization. Breakthroughs
in functionalization strategies for enhancing the dispersion of CNTs in aqueous and non-aqueous
solvents are carried out. This also reduces toxicity and enhances the bio-distribution. CuI has
lately been used as a catalyst in process of direct amidation on CNTs, while Cu2+ and Ni2+ have
undesirable consequences in comparison with Cu1+ salts. Copper-catalyzed amidation provides
surface polyamine groups that, in return, demonstrated excellent aqueous dispersibility. There
have been several other methods to enhance the solubility suchlike esterification, oxidation,
functionalization by cycloaddition reaction, etc.
2.6.2. Challenge in reproduction of identical CNTs
The morphology, content, and structure of impurities in as-prepared CNT samples seem to be
well recognized to significantly rely on their methods and conditions of synthesis. There is thus
no standardized system for the consistently production of high-quality CNTs with identical
properties. These also depends on the varying working conditions utilized during the synthesis
and purification of nanotubes i.e., temperature, pressure, catalysts used, surrounding conditions
of chamber, transition metals, etc. Even a certain variation in these factors can significantly
changes the chemical and structural properties of CNTs produced.
2.6.3. High production cost
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Fortunately, significant advances have been made in recent years in the purification of CNTs,
and small quantities of high-purity CNTs can indeed be easily acquired considering the
drawbacks of high costs and long-term involvement. A study has been conducted by Ebbesen et
al., a gas phase purification to open and purify MWCNTs by oxidizing the as-prepared sample in
air for 30 min at 750 °C. However, after the above purification, only a small amount of pure
MWCNTs (1–2% wt.) remained. The impurities present in CNTs are produced due synthesis
techniques and hence, purification is mandatory to get rid of carbonaceous matters, catalyst,
metal impurities. So, to obtain CNTs of high quality, the yield reduces to a significantly limited
amount further leading to high cost of production. Thus, it is very difficult to maintain the high
quality, lesser impurities, high yield and economical CNT production all at the same time and
this seems a major limitation. Setting up a standard for CNT purity evaluation is important to
determine and enhance purification validity. The use of effective CNT purification techniques
that can achieve high-quality and homogeneous CNTs without doubt would significantly
expedite fundamental research or even practical applications of CNTs.
https://www.sciencedirect.com/science/article/pii/S1773224720303750
2.6.4. High energy desirable
The graphite rods were exposed to a very high temperature and pressure and high dc voltage is
also applied so that the graphite rods get evaporated to form nanotubes and get deposited onto
the anodes in the heating chamber. The high temperature required for arc-discharge and same for
laser ablation technique significantly greater than those of other CNT production processes. The
major drawback for this method being that for CNT synthesis, it utilizes higher temperatures,
usually causing the expansion of CNTs with fewer structural defects compared to several other
methods; consequently, there seems to be comparatively limited influence over the orientation
(i.e., chirality) of the nanotubes formed, which would be essential for structural function and
characterization.
2.7.
Conclusion of neuroregeneration and neuroprotection
Neuroprotection and neuroregeneration are the subject of a vast and dynamic field of research.
The elderly population is the main target in the case of neurodegeneration, with ageing of the
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
brain as a principal factor. Study of the cellular and molecular mechanisms involved in the
neurodegeneration and neuroregeneration of the ageing brain could unmask new therapeutic
approaches to reduce the degradation of neurons. Successful management of AD and PD may be
achieved by developing novel and efficient therapies that sustain the self-repair ability of the
brain. On the basis of the studies reported in the literature, a wide range of nanotechnologies has
been designed to assess the functional outcomes of CNTs when used as scaffolds to provide
mechanical support for neural tissues, and to deliver drugs, nerve growth factors, antibodies, and
proteins to a particular area of the brain in AD and PD to stimulate regeneration of neurons.
Nano-technological solutions based on CNT could be expensive to implement for
neuroprotection and neuroregeneration, so researchers should be looking at the cost-effectiveness
of treatments for neural disorders that involve use of CNT.
Some in vivo studies have assessed the toxic effects of CNTs that have accidentally penetrated
the body and probably translocated in the CNS. CNT have been reported to have toxic effects on
dorsal root ganglion neurons and to induce membrane damage. In addition to these limitations,
use of CNT is steadily increasing worldwide. The potentially toxic outcomes of using CNT need
to be studied in depth in mouse hippocampal neuron models before we embark on clinical trials,
to prevent adverse clinical and environmental effects.
Functionalized CNTs provide enhanced solubility, and improve biocompatibility and mechanical
properties. The improved cytocompatibility of CNTs after functionalization is shown in Figure
17. Toxicity issues must be overcome to fulfill the promise of CNTs for nanotechnological
application in neuroscience. Further research may show that the effectiveness of
nanotechnologies can outweigh their risks, and the next decade will present huge scope for
developing and delivering technologies in the field of neuroscience.
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Carbon nanotubes as emerging candidates in neuroregeneration and in neurodrug delivery
Figure 17. Improved cytocompatibility of carbon nanotube after functionalization.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495782/#!po=65.4762
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