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2.1 CARBON NANOTUBES

CNTs are allotropes of carbon, made of graphite with a cylindrical nanostructure several
micrometers in length and nanometer scale in diameter and discovered in 1991 by a Japanese
scientist S. Lijima. They are either made up of single-walled or multi-walled graphite layers
and are therefore called single-walled carbon nanotubes (SWCNTs) or multi-walled carbon
nanotubes (MWCNTs) . Their impressive structural, mechanical and electronic properties are
due to their small size, their incredible mechanical strength and their high electrical and
thermal conductivity. Firstly, they were used as additives to various structural materials for
electronics, optics, plastics, and other materials of nanotechnology fields. Since the beginning
of 21st century, they have been introduced in pharmacy and medicine for drug delivery system
in therapeutics. Recently, CNTs have attracted focus due to possible applications as a drug
delivery vehicle or “nanocarriers” in cancer therapy and other areas of medicine.[73]
CNTs have potential advantages over other drug delivery systems; these include their ability to
carry high cargo loading, intrinsic stability, and structural flexibility, which prolong time of
circulation and bioavailability of carried drug molecule[68]. They have been proven an
excellent vehicle for drug delivery by penetrating into the cells directly and keeping the drug
intact without metabolism during transport in the body . Many studies have demonstrated that
when bonded to CNTs, the drug molecules are delivered more effectively and safely into cells
than by traditional methods. This fantastic discovery has opened a new way for drug
preparations that is completely different from traditional techniques used in pharmaceutical
industry and radically changed anterior concepts of pharmacology .[74]

It has been first applied to bind antineoplastic and antibiotic drugs to carbon nanotubes for
cancer and infection treatments respectively. Then, linkage of biomolecules (gene, proteins,
DNA, antibodies, vaccines, biosensors, cells, etc.) to CNTs has been also reported for gene
therapy, immunotherapy, tissue regeneration and diagnosis of different ailments. Therefore, in
a very short time, CNTs have become the focus of attention by scientists in a wide variety of
disciplines. Besides these main applications of CNTs, they have been shown as a powerful
tool for enantiomer separation of chiral drugs and chemicals in pharmaceutical industry as well
as in laboratory and for extraction of drugs and pollutants in different media by solid phase
extraction before analysis. The therapeutic effect of drug is increased but the toxicity
associated with this also increases.[75]

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Fig 2.1 Diagrammatic representation and electron microscopy images of SWCNTs and MWCNTs.

(A) to (D): SWCNTs; (E) to (H): MWCNTs. Scanning electron microscope (SEM) images
show SWCNT (B) and MWCNT (F) aggregates; transmission electron microscope (TEM)
images show raw SWCNT bundles (ropes) with metal nanoparticles (C), and individual
multiwall tubes (G). High-resolution TEM images show a cross-section of a SWCNT bundle
(D) consisting of >25 tubes and some amorphous carbon on the edges, and a longitudinal

cross-section of a MWCNT (H) with an empty central cavity and ̴ 20 walls on each side and
some amorphous carbon. [76]

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2.1.2 Role of CNTs in drug delivery

Ever since the discovery of CNTs in 1991 by S. Lijima, CNTs are widely used as a drug
carrier for anticancer drugs. The chemotherapeutic agents are always associated with severe,
sometimes fatal, toxicity due to lack of target specificity.[77] Several attempts have been
made to reduce side effects for example by liposomal encapsulation of afatinib. To overcome
these side effects CNTs as drug delivery carrier is taken into consideration. CNTs show
advantages over other nano-sized delivery systems, such as an exceptionally high drug
loading capacity due to their high surface area and possibility for incorporating additional
therapeutic and diagnostic moieties, either on surface or on their inner cavity. In addition,
they interact with cellular membranes in a unique way: some CNTs have been reported to
enter mammalian cells by an endocytosis- independent, "needle like" penetration mechanism,
which allows direct cytoplasmic delivery of therapeutic drugs. [78]

A number of studies have reported successful delivery of anti- cancer drugs to human cancer
cells by means of carbon nanotubes. Several cell types can uptake CNT, including cells of
the immune system, such as macrophages, monocytes, natural killer (NK), dendritic cells, T
and B cells. CNT were shown to activate cells from the innate immune system, such as
monocytes, macrophages and dendritic cells. Both functionalized and non-functionalized
CNTs activate several genes involved in monocyte response to infection or vaccination, such
as nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB), interleukin-1β
(IL-1β), IL-6, tumor necrosis factor-α (TNF-α), among others in a human monocytic cell line,
THP-1 cells. [79]

However, the non-functionalized CNTs also increased the expression of genes related to
oxidative stress and apoptosis . The functionalized CNTs (9.5 nm and 30 nm diameter) were
oxidized and further modified to incorporate ammonium; both versions with and without
ammonium were studied. These nanotubes were shown to be non-toxic to both THP-1 cells
and human primary monocytes and induced production of cytokines and chemokines like IL-
1β, IL-6, TNF-α and IL-10). These mediators are involved in processes like recruitment of T
cells to infection sites and inflammation. These studies suggest that CNTs can induce an
innate immune response dependent both on their functionalization type and diameter.[80]
Gene delivery shows great potential as a method of treating certain diseases, it could benefit
from the use of CNT vectors to solve a major difficulty, intracellular transport

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DNA degrades rapidly outside the nucleus due to enzymatic action in the cytoplasm, and thus
it is necessary to associate it with a vector to assist the gene in its delivery to the nucleus Viral
vectors have been used extensively in gene delivery due to their natural ability to invade cells.
However, they can often trigger an unwanted immune response . Since CNTs can bind to
various molecules to augment cell interactions, they present a novel solution to the problem of
finding a suitable gene delivery vector .[81]

2.1.3 Delivery of anticancer drugs

CNTs are an attractive drug delivery carrier for anticancer compounds. The conventional
methods for cancer treatment such as surgery, has risks while operating near or on vital body
organs. Also, treatment with chemotherapy and radiation therapy suffer from a limitation.
The use of CNTs as drug delivery carrier has following major benefits:

 Less quantity of drug is required for delivery and results in more economical
treatment.
 Lower concentration of cytotoxic compounds are delivered to non-target sites.

 Wide range of compounds can be attached for variety of purposes including;

therapeutic, targeting and diagnostics.[82]

2.2 Examples of some anticancer drugs delivered by carbon nanotubes:

1. Afatinib.
It is in a class of medication called kinase inhibitors . It is widely used in treating various
kinds of cancers. Traditionally, afatinib is administered intravenously, resulting in its
inefficient distribution, low selectivity and inability to cross the cellular barriers. These
limitations of traditional administration of doxorubicin can be counteracted by using CNTs as a
novel drug delivery carrier, due to their capability of immobilizing therapeutic molecules on
the surface or in their hollow space and transporting them through mammalian cell
membranes .[83] Afatinib can be loaded on CNT via various approaches. In one approach,
afatinib can be loaded on polysaccharide materials (sodium alginate) coated carboxyl-
functionalized CNT.Afatinib binds to CNT at pH 7.4 and gets released at lower pH, which is
characteristic of certain tumor environments.[84]

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2. Platinum-based anticancer drugs

Cisplatin is a platinum based anticancer drug commonly used to treat various types of cancers.
It triggers apoptosis by binding to DNA in vivo to induce DNA crosslinking. However, a
number of undesirable side effects limits its application. CNT-based drug delivery can
overcome these limitations by protecting light sensitive cisplatin from external reactive
species. Cisplatin can be encapsulated inside tip opened and shortened SWCNTs, which are
treated with strong acid and annealed in a high vacuum environment. However, the effect of
released cisplatin from SWCNT is not greater than of bare cisplatin, which may be attributed
to the loss of cisplatin’s activity during encapsulation .[85]

Similarly, another platinum based anticancer drug, carboplatin, can be incorporated inside
CNTs and the effectiveness of drug filled CNTs on growth of cancer cells was studied
(Hampel et al; 2008). Carboplatin retains its structure inside CNTs and effectively suppresses
the growth of bladder cancer cells, whereas CNTs per se do not influence the growth of tumor
cells, thus confirming the absence of any intrinsic cytotoxicity .[86]

3. Other anticancer drugs

A DNA topoisomerase-1 inhibitor, 10-hydroxycamptothecin, has potent antitumor activity. It

was linked covalently to MWCNTs using diamino-triethylene glycol as a hydrophilic spacer
which exhibited better in vitro and in vivo anticancer activity than the marketed formulation.
The formulation also had a relatively longer blood circulation apart from high concentration
at tumor sites. Methotrexate (MTX), a folate antimetabolite antitumor drug was observed to
enter rapidly in Jurkat cells when administered as MTX-CNT complex
Hexamethylmelamine, an antitumor agent, can be incorporated inside C60 capped
SWCNT/double wall carbon nanotubes (DWCNT).[87]

2.3 Delivery of other drugs

A part from anticancer drugs, CNT-based drug delivery systems have also been employed for
the delivery of other drugs. Dapsone, an anti-microbial and anti-inflammatory drug, was
modified onto functionalized-MWCNTs. There was non-obvious apoptosis of rat potential
macrophages when dapsone-CNT or oxidized CNTs, up to 50µg/ml was used. Higher levels of
both types of CNTs induced apoptosis, which was greater in the case of oxidised-CNTs.

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However, prolonged incubation of cells (>3 days) in 50µg/ml of dapsone-CNTs triggered

apoptosis. Similar levels of individual dapsone and oxidised-CNTs caused oxidative stress,
whereas dapsone-CNTs do not cause any oxidative stress. Therefore, dapsone-CNTs can be
effectively used for treating dapsone-sensitive intracellular microorganisms and dapsone-
responsive inflammatory diseases .[88]

Dexamethasone is a widely used anti-inflammatory and immunosuppressant drug for treating

many inflammatory and autoimmune diseases. Chitosan-SWCNTs can be used as host carrier
films for electrically stimulated delivery of dexamethasone. An accelerated cellular uptake
and a complete drug release was obtained due to electrostatic repulsions of SWCNTs and
dexamethasone when -0.8V vs. Ag/AgCl was applied. The passive release of dexa-
methasone, i.e. without any stimulation, decreased by the addition of SWCNTs, due to
possible attractive interactions between the drug and SWCNTs. The application of a positive
potential (+0.15 V vs. Ag/AgCl) to the Chitosan-CNT- dexamethasone composite decreased
the release of dexamethasone .[89]

Ketoprofen, one of the non-steroidal anti-inflammatory drugs with analgesic and antipyretic
effects, inhibits the production of prostaglandin in the body. It is a drug of common
prescription for treatment of inflammatory conditions due to arthritis or severe toothaches
caused by gum inflammation. An electro sensitive transdermal DDS, composed of a polymer
matrix and MWCNT, increased amount of drug released with enhanced potentials, which can
be attributed to high electrical conductivity of CNTs.[90]

Amphotericin B, a polyene antifungal drug, is often administered intravenously for the

treatment of systemic fungal infections. However this drug had serious and potential lethal
side effects to mammalian cells. The toxicity of this drug was may be due to its low water
solubility that results in the formation of aggregates (Szlinder-Richert et al., 2004). The
binding of Amphotericin B to f-CNT increased its solubility and prevent its aggregation. Also,
the drug efficacy was improved and the antibiotic activity was modulated. F-MWCNTs was
used for targeted delivery of Amphotericin B MWCNTs was treated with acid to induce
carboxylic groups and then functionalized with two orthogonally-protected amino acids.[91]
Fluorescein isothiocynate (FITC) and Amphotericin B was conjugated to f-MWCNT.
Amphotericin B preserved its high antifungal activity even after binding to MWCNT and the
Amphotericin B-CNT complex was transported across the mammalian cells without causing

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any cytotoxicity . Theophylline was encapsulated in a CNT-filled alginate (AL) microsphere.

The drug leakage decreases when AL/CNT microsphere was used in comparison to AL
microsphere. The AL/CNT microsphere inherits the pH sensitivity of the AL microsphere and
had a more sustainable drug release profile.However, the cytotoxicity of AL/CNT microsphere
was similar to that of AL microsphere .[92]

Acetylcholine (Ach) is an important neurotransmitter in the peripheral and central nervous

system in many organisms including humans. The delivery of Ach into the brain is useful for
the treatment of Alzheimer’s disease. Lysosomes and mitochondria was identified as the
pharmacological and toxicological target organelles, respectively for SWCNTs (Yang et al.,
2010). Therefore, SWCNTs were utilized to release Ach into the brain for treating the
experimentally induced Alzheimer’s disease with a moderate safety range. This was done by
precisely controlling the doses, ensuring that SWCNTs preferentially enter lysosomes but not
mitochondria .[93]

Carvedilol, an antihypertensive drug is a poorly water soluble drug. Various attempts had been
made to load carvedilol on MWCNT-COOH, where more carvedilol was found inside the
carboxyl-functionalized MWCNTs using the solvent method. When carvedilol loaded on f-
MWCNTs than in MWCNTs, the solubility was increased further, thereby indicating an
improvement in its biocompatibility .[94]

Delivery of biomolecules

a. DNA and RNA

DNA can be attached to the amino groups of f-MWCNT. The linkage of DNA to f-MWCNT
is utilized for improving nanotubes’ dispersibility in aqueous media as well as for efficient
gene transfection without the use of viral genes. MWCNTs functionalized with cationic
polyelectrolyte were used for the intracellular delivery of antisense oligo-deoxy-nucleotides.

DNA binds to SWCNTs and can be effectively released into HeLa cells by the cleavage of a
disulfide bond between f-SWCNT and DNA in the cytosol followed by its nuclear
translocation. The transportation of DNA by SWCNTs inside the two cell lines, i.e. adherent
HeLa and non-adherent HL60 cells, suggested internalization by energy-dependent
endocytosis
.[95]
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SWCNTs have been advocated as carriers for the intracellular delivery of ssDNA probe. This
strategy can avoid nuclease digestion or protein interaction, thus improving the efficiency of
transfection. The binding of DNA probes to SWCNTs protect them from enzymatic cleavage
and disturbance from nucleic acid binding proteins. SWCNT-bound DNA probes, which bind
to a specific target mRNA, were found to improve self-delivery and intercellular biostability in
comparison to free DNA probes .[96]

b. Proteins
Bovine serum albumin (BSA) can be bound covalently to SWCNT/MWCNT by diimide-
activated amidation to form CNT-BSA conjugates with high water solubility about 90% of
BSA molecules retain their activity, as determined by the total protein micro-determination
assay. The noncovalent and nonspecific binding of various types of proteins (molecular
weight<80kD) to the sidewalls of SWCNT was reported. The protein transport and its uptake
through CNT carriers were generic for varied adherent and nonadherent cell lines and
internalized by energy–dependent endocytosis .[97]

In vivo biodistribution
Biodistribution is an important parameter to evaluate the biosafety of nanomaterials. In vivo
biodistribution of radiolabeled I-SWCNTs follows following order with decreasing
concentration at organ: trachea > urine>stomach > intestine > serum > bladder> bloodvessel>
kidney > liver > lung > adrenal > femur > spleen > testis >thymus > thyroid > heart > fat >
muscle > brain. Most of MWCNTs were accumulated in the lungs, with just a small amount
of retention in the liver and spleen and could be eliminated slowly from the tissues. The
highest uptake could be observed in the liver, lungs, and spleen within 24 h, which increases
slowly with time at 8, 16, 24 h after administration .[98]

2.4 Cellular uptake of carbon nanotubes

Most cancer drugs works intracellularly, this means that the drug must be taken up by the cell to cause
the desired effect. To engineer the nanoparticles, it is important to have detailed knowledge of the
internalization mechanisms and the intracellular routing of the drug and nanoparticles. While ions,
small proteins and molecules such as glucose and amino acids are transported through the membrane by
specialized transporter proteins, larger substances such as nanoparticles are too big for the specialized
transporter proteins and are internalized through endocytosis. Endocytosis involves folding the plasma
membrane into vesicles that are released from the membrane, and transports its content into the cell.

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The opposite, where intracellular vesicles fuse with the cell membrane to release the contents
extracellularly, is termed exocytosis. Endo- and exocytosis are important for many of the cells vital
processes such as uptake of nutrients, transcellular transport, signal transduction and phagocytosis.
Also, this process is important for transporting membrane bound proteins such as receptors, transporter

proteins and signaling molecules .[99]

Figure 2.2: Cellular uptake of nanoparticles via different endocytotic pathways. The pathways differ in
size and mechanism of vesicle formation.[100]

CNTs are too big for the specialized transporter proteins and are internalized through
endocytosis. Endocytosis is divided into two subgroups, phagocytosis and pinocytosis .
Phagocytosis is mainly performed by professional phagocytes; macrophages, monocytes
neutrophils and dendritic cells.[101]

These are all part of innate immune system where phagocytes can internalize whole bacteria
and cells with a size up to tens of micrometers. Phagocytosis occurs only after the object has
been opsonized, that is the cell/bacteria/particle has been covered by proteins such as
immunoglobulins and complement components. These proteins are recognized by the
phagocytes prior to phagocytosis. Cancer cells would internalize nanoparticles through
pinocytosis. Pinocytosis can further be divided into subgroups as:[102]

 Clathrin mediated endocytosis

 Caveolae mediated endocytosis

 Clathrin and caveolae independent endocytosis.

 Macropinocytosis.

2.5 Clathrin mediated endocytosis

This is the most important mechanism for internalization of nutrients and macromolecules. It
is a receptor mediated mechanism in which substrate to be internalized must bind to a surface
receptor. Multiple receptors bind to bigger objects and create a concentrated area with
receptors. These areas are covered by an adaptor complex AP2 and clathrin, a triskelia- shaped
macromolecule, on the cytosolic side. Clathrin and AP2 form a curvature in the membrane,
which continues with further addition of clathrin and AP2. Finally, a vesicle is formed which
is cut off from the membrane by a complex of dynamin proteins.[103]

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2.6 Caveolin-mediated endocytosis

After clathrin-mediated endocytosis, caveolin-mediated endocytosis is the main mechanism of

endocytosis. Caveolin-oligomerization in the plasma membrane creates an invagination
forming a flask-shaped vesicle. This vesicle is cut off from the membrane by dynamin2. Only
smaller volumes and particles with a diameter around 60 nm are captured by this mechanism.
However, for beeds above 200 nm, it has been reported that caveolin is involved in uptake
superior to clathrin-mediated endocytosis.[104]

Figure 2.3: The internalization via clathrin mediated endocytosis. The internalization of
receptor- bound molecules is exemplified by transferrin and the transferrin receptor.
Adaptor complex 2 and clathrin cover the cytosolic side of the cell membrane, and creates
the invagination. A dynamin complex then assembles at the neck of the forming vesicle
and cuts it down from the plasma membrane .[105]

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2.7 Clathrin and caveolin independent endocytosis

Cellular uptake can also occur through macropinocytosis, which are large ruffles in the cell
membrane folding over to create a vesicle. This mechanism is not selective, meaning that it
does not depend on receptors or stimuli from the internalized material. Macropinocytosis also
occurs at a relatively constant rate in most cells and serves as a method for sampling the
environment. This method includes phagocytosis, macropinocytosis and transcytotic process
.[106]

2.8 Intracellular route

After internalization, the newly formed vesicle will fuse with hydrolase containing vesicles
from the Golgi complex to create early endosomes. This process is rapid, the vesicle is
typically formed within 2 minutes and fusing with vesicles from Golgi will occur for
approximately 10 minutes. The early endosome become late endosomes when pH is dropped

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towards 5 and they lose the ability to fuse with endocytic vesicles. After 10-40 minutes, the
late endosomes fuse with lysosomes. After being degraded in the lysosomes, the material can
either be removed from the cell by exocytosis, delivered to specific organelles or be released
in cytosol (Fig.2.4). Some nanoparticles also go to nucleus or cytoplasm and don’t follow the
lysosomal route.[107]

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Fig 2.4: The uptake and possible intracellular routes of nanoparticles .[108]

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2.9 Cytotoxicity
The cytotoxicity of CNTs needs to be extensively investigated in vitro and in vivo if they are
employed as drug carriers. Numbers of research reports have focused on this issue, but the
reported cytotoxicity findings of CNTs are incompatible with each other. These conflicting
reports had attributed to variability in the doses, properties, purification and functionalization
of CNTs employed for various cytotoxicity studies. Carbon nanotubes had developed into
new materials with a variety of industrial and commercial applications. In contrast, the
physiochemical properties of CNT at the nanoscale render them the potency to generate toxic
effects. Indeed, the potential health impacts of CNT have drawn a great deal of attention in

recent years, Owing to their identified toxicological and pathological consequences including
cytotoxicity, inflammation, fibroisis, genotoxicity, tumorigenesis and immunotoxicity.
Understanding the mechanisms by which CNTs induce toxicity and pathology is thus urgently
needed for accurate risk assessment of CNT exposure in humans and for safe and responsible
development and commercialization of nanotechnology.[109] Nanotubes have unique physical
and chemical characteristics, because of which their use has increased greatly in the field of
medicines as a drug carrier. But with increased use, the concern on their environmental and
health effects is growing rapidly. The effect of CNT on mammalian cells has been reported in
multiple cell types, which provides guidance for screening and mechanistic and in vivo
investigations on CNT toxicity. CNT conferred cytoxicity in a dose and time dependent
manner in different cell types .Various reports on their toxicity had reported till now. Reported
the pulmonary toxicity of SWNTs in mice. It was also found that SWNTs induce dose-
dependent interstitial granulomas.[110]

Both SWCNT and MWCNT induced secretion of inflammatory cytokines, chemokines, and growth
factors such as tumor necrosis factor, interleukin monocyte chemotactic protein and transforming
growth factor in mouse RAW 264.7 macrophages, indicating that CNT have the potential to trigger
inflammatory responses. CNT treated cells demonstrated elevated levels of intracellular reactive oxygen
species (ROS) in a variety of cell types, which leads to oxidative stress and toxicity. In addition, CNT
generate genotoxic effects in cultured cells, such as DNA strand breakage, DNA base oxidation,
formation of micronuclei, and chromosomal aberrations, which were recently reviewed by these genotoxic
effects of CNT suggest a potential of CNT to cause cancer in animals and humans.[111].

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2.9.1 Mechanisms of cytotoxicity

Several cytoxicity mechanisms have been proposed with some claiming the cytotoxicity of
CNTs due to the disruption of intracellular metabolic pathways, and others stating that CNTs
causes oxidative stress and membrane damage. The most developed pattern for determining the
effect of CNTs on the mammalian cells is the generation of reactive oxygen species
(ROS) due to oxidative stress.
Although the mechanisms underlying cytotoxicity are not fully understood, accumulation of
reactive oxygen species (ROS) and oxidative damage is likely a contributing factor So the
mechanisms of cytotoxicity can be categorized as:

• Physical damage to the cells.

• By activation of signaling mechanisms intracellularly .[112]

Insertion of CNT in lipid bilayer resulted in alterations in lipid bilayer organization and leads
to increased diffusion of low atomic weight ions and small molecules. Simulation studies
suggest that insertion of CNTs in plasma membrane results in formation of pores through
which solvated ions can pass. Experimental studies using liposomes and RAW 264.7 cells also
confirmed membrane damaging potential of MWCNTs. The disruption of plasma membrane
receptor MARCO and disrupt plasma membrane.[113]

The role of oxidative stress has been highlighted in several studies. Although it is generally
believed that oxidative stress generated due to CNT exposure may result in cell death, the
enhanced production of free radicals may not necessarily lead to cell death. Further, treatment
with antioxidants failed to abrogate toxicity of MWCNTs in J774.1 cells. Contrary to the in
vitro and in vivo studies, MWCNTs did not generate free radicals but may act as free radical
scavengers. Oxidative stress may lead to activation of other pathways and DNA damage which
may drive the cell machinery to apoptosis. Apoptosis is an important mode of cell death in
response to CNT exposure. One of the important reasons for in vitro reduction in cell viability
and in vivo toxicity appears to be apoptosis. Poly (ADP) ribose polymerase (PARP) activation
was observed in response to MWCNTs. The SWCNTs did not induced apoptosis after
intravenous administration in mice or in J774.1 cells incubated with MWCNTs. The role of
necrosis in cell death had also been implicated. Cell cycle arrest had also been demonstrated
after incubation with SWNTs .[114]

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Incubation of SWCNTs and MWCNTs with mesothelial cells showed an increase in histone
2AX phosphorylation and activation of ERK, JNK, and p38. Further, AP-1, NF-kB and Akt
activation was also observed after incubation of cells with SWCNTs. The activation of NF- kB
and ROS production may lead to activation of NF-kB and could account for toxicity of CNTs.
Further, CNTs may also lead to altered expression of cytoskeletal and cell adhesion proteins.
The role of inflammation in tissue damage has also been observed. Inflammation may arise
either due to oxidative stress or physical injury induced by CNTs. Alternatively, the activation
of intracellular signaling proteins like NF-kB .[115]

Fig 2.5 Prooxidant pathway for NP-induced toxicity .[116]

Various NP exhibit oxidative stress dependent toxicity. Upon CNT exposure, ROS generation is
capable of inducing oxidative DNA damage, strand breaks, protein denaturation, and lipid peroxidation
thereby demonstrating the mutagenic and carcinogenic characteristics associated with CNT. Excess free
radical production leads to mitochondrial membrane damage causing necrosis and cell death.
Phagocytes including neutrophils and macrophages generate massive ROS upon incomplete
Phagocytosis of CNT through the NADPH-oxidase enzyme system whereas CNT-induced ROS triggers
an inflammatory cascade of chemikine and cytokine

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expression via activation of cell signaling pathways such as MAPK, Akt and receptor tyrosine
kinase. Furthermore, oxidative stress mediated stimulation of these cellular mechanisms
results in transcription of genes responsible for fibrosis, epithelial-mesenchymal transition and
carcinogenesis. CNT-elicited ROS is at the center stage for majority of the ensuing adverse
outcomes.[117]

2.9.2 CNT-induced oxidative stress

One of the most frequently reported toxicity endpoints for CNT is the formation of ROS

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which can be either protective or harmful during biological interactions. Oxidative stress may
be caused directly by CNT-induced ROS in the vicinity or inside the cell or could arise more
indirectly due to effects of internalized CNT on mitochondrial respiration or in depletion of
antioxidant species within the cell.[118] Moreover, NADPH-mediated ROS are critical for
SWCNT-induced pulmonary responses. The most likely mechanism for CNT- induced
oxidative stress and lung toxicity involves mitochondrial dysfunction. Incomplete
Phagocytosis of CNT, presence of transition metals and specific reactive groups on CNT
surface are key drivers of ROS generation. Metal impurities such as Fe, Co, and Ni introduced
within the CNT during their synthesis are key factors driving CNT-mediated ROS response
(Goff et al., 2011). CNT-induced oxidative stress mediates important cellular processes
including inflammation, cell injury, apoptosis, and activation of cellular signaling pathways
such as MAPK and NF-kB, which were implicated in the pathogenesis of lung fibrosis
(Bonner et al., 2002). For instance, SWCNT dependent hydroxyl free radical generation leads
to activation of molecular pathways MAPK, AP-1, NF-kB, and Akt associated with cell
proliferation and tumor progression in vitro. Several studies demonstrate SWCNT-induced
oxidative stress. Similarly, MWCNT exposures had reported to induce ROS both in vitro and
in vivo. Interestingly, oxidative stress was reported to be a mechanism for biodegradation of
CNT. SWCNT undergoes oxidative biodegradation via myeloperoxidase, a prooxidant enzyme
involved in host defense responses.[119]

2.10 Role of ROS in CNT-induced inflammation

ROS and inflammation demonstrate an interdependent relationship in the case of exposure to
CNT. Inflammatory cells such as macrophages and neutrophils induce enormous ROS release
in order to get rid of the CNTs. However, CNT exposure mediated oxidative stress leads to
activation of RTK, MAPK, Akt, and NF-𝜅B contributing to the proinflammatory cascade
Accordingly, CNT-induced ROS were reported to elicit pro- inflammatory transcription
factors such as NF-𝜅B, AP-1 and MAPK in vivo. This was found to be an inflammation
dependent response MWCNT treatment in macrophages mediates ROS-dependent activation
of NF-𝜅B pathway, thereby inducing the expression of chemokines and cytokines such as
TNF-𝛼, IL-1𝛽, IL-6, IL-10, and MCP-1. Likewise, MWCNT-induced nitrosative stress in vivo
is associated with pulmonary inflammation .[120]

2.11 Role of ROS in CNT-induced genotoxicity

CNT elicit genotoxic effects through direct interactions with DNA or indirectly via CNT-
induced oxidative stress and inflammatory responses. CNT-induced sustained oxidative stress

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can result in DNA damage and abnormal cell growth, possibly leading to carcinogenesis and
fibrogenesis .A plethora of studies demonstrate the genotoxic potential of both MWCNT and
SWCNT. ROS can activate cellular signaling pathways resulting in cell cycle arrest and
apoptosis. CNT induce a multitude of genotoxic responses including DNA strand breakage,
oxidation, micronuclei induction, chromosomal aberrations, formation of
𝛾H2AX foci, and mutant frequencie. Oxidative stress-dependent DNA breakage and repair and
activation of signaling pathways including poly-ADP-ribose polymerase (PARP), AP-1,
NF-𝜅B, p38, and Akt were reported in human mesothelial cells exposed to SWCNT. CNT
induce ROS dependent lipid peroxidation both in vitro and in vivo. A number of studies account
for mitochondrial membrane depolarization, damage, and oxidative stress upon CNT exposure.
Unlike the traditional Prooxidant effect of NP, CNT had reported to sequester ROS which in turn
is associated with their structural defects. This quenching was reported to be related to the
genotoxic and inflammatory effects observed with CNT.[121]

2.12 Role of ROS in CNT-induced fibrosis

Increased ROS had implicated in lung inflammation and fibrosis. The inflammatory cascade
was reported to contribute to oxidative stress mediated lung injury. Exposure to CNT results in
expression of genes responsible for inflammation and fibrosis via the activation of cell
signaling pathways and transcription factors including NF-𝜅B, STAT-1, MAPK, and RTK.
ROS-dependent p38-MAPK had shown to be responsible for CNT- induced collagen and
angiogenic responses. Additionally, SWCNT induce fibrogenic effects via ROS-mediated NF-
𝜅B activation. whereas MWCNT induce fibroblast to myofibroblast differentiation via ROS-
dependent NF-𝜅B activation . Insertion of CNT in lipid bilayer resulted in alterations in lipid
bilayer organization and leads to increased diffusion of low atomic weight ions and small
molecules. Experimental studies using liposomes and RAW264.7 cells confirmed membrane
damaging potential of MWCNTs. The MWCNTs may bind to membrane receptor MARCO
and disrupt plasma membrane.[122]

Role of oxidative stress had highlighted in several studies and was believed that oxidative
stress generated due to CNT exposure may results in cell death. Also, oxidative stress may
lead to activation of other pathways and DNA damage which may drive the cell machinery to
apoptosis. Apoptosis is an important mode of cell death in response to CNT exposure.

The CNTs may also results in activation of several stress pathways. The activation of
transcription factors like NF-kB and AP-1 lead to production of inflammatory cytokines and
could account for toxicity of CNTs .[123]
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