Abstract SWCNTs can be successfully used as

Abstract

 

Single-wall carbon nanotubes have unique
physiochemical properties such as high drug loading efficiency, larger surface,
high thermal conductivity, which make them attractive in the range of
biomedical applications, particularly in anticancer drug delivery. However,
toxicity issues associated with CNTs have been raised. The cytotoxic profile of
the CNTs have not been fully understood yet and the available experimental data
related to CNT’s toxicity is also inconsistent. Hence, in this study, I will
evaluate the cytotoxicity effects of pristine and
functionalized single-wall carbon nanotubes (PEG-SWCNT, COOH-SWCNT,
Trion-X-100-SWCNT and PAA-SWCNT) on Widr colon cancer cells. Firstly, SWCNTs
will be purified and functionalized and then both pristine and functionalized
SWCNTs (f-SWCNT) will be characterized by using FTIR, DSC and TEM. The
cytotoxic responses of these f-SWCNT and pristine CNTs will be analysed using neutral
red uptake and MTT assays, Moreover, morphological analysis, mitochondrial
membrane potential, reactive oxygen species will be carried out in this study. The
hypothesis of this study is to observe the low toxicity effects from
functionalized CNTs compared to the pristine CNTs. This study will help to
understand the effects of functionalization on CNT toxicity. Most importantly,
functionalized SWCNTs can be successfully used as an anticancer drug delivery
system to deliver drugs to the target sites without having a risk of
aggregation in the body. Furthermore, health side effects associated with
pristine CNTs can also be prevented from using f-SWCNT.

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1.0 Introduction

 

Cancer is
responsible for the highest number of deaths in modern society next to the
heart and cerebrovascular diseases. According to WHO, cancer cause
approximately 8 million deaths annually all over the world. Most of the current
cancer treatments engage with the chemotherapeutic agents which have significant
toxicity because of its low target specificity (Kushwaha et al. 2013). As the result of the rapid
progress of nanotechnology, these conventional strategies started to replace
with some promising and innovative alternatives such as nanoscale drug delivery
systems. Among these platforms, carbon nanotubes (CNTs) have received a
significant attention in the last two decades due to its unique physiochemical
properties (Sanginario, Miccoli & Demarchi 2017).

CNTs
are cylindrical nanostructures which made of the sp2 hybridized
carbon atoms in a hexagonal arrangement. They can be categorized into two main
subgroups namely single-wall CNTs (SWCNTs) and multiwalled CNTs (MWCNTs)
depending on the number of graphite sheets that they constitute. Nanotube which
is formed by rolling up a single graphene sheet, is defined as SWCNT and those
which are formed by rapping more than one sheet in a concentric fashion are
known as MWCNTs (Kushwaha et al. 2013).
CNTs are capped at both ends of the carbon tube with a hemispherical
arrangement of a backyball structure. Furthermore, SWCNTs
have attracted considerable interest over the most of metal nanoparticles due
to their properties such as intrinsic stability, high encapsulation efficiency,
prolong circulation time, and etc (Singh et al. 2006; Wang
et al. 2004). Therefore, SWCNT
will be used in this study with an average diameter of 0.4-3 nm and an average
length of 1-5 ?m (Kushwaha et al.,
2013)

Several methods have been employed to
synthesize SWCNTs such as Arc discharge method, laser ablation method and
chemical vapour deposition (CVD) method. 
The Arc discharge method is one of the common technique which applies
the high DC current between two purity graphite electrodes to produce carbon deposit
at the cathode (Toma 2009). This method requires the further purification to
obtain pure and efficient SWCNTs (Edgington 2011).

Laser
ablation is the second most popular method to synthesis SWCNTs. In this method,
metal-graphite target is placed inside a tube, within a high-temperature
furnace and laser beam is scanned across the target surface to vaporize the
graphite target. Carbon nanostructures will be deposited outside of the furnace
on the water-cooled copper collector (Szabo et
al. 2010). This method facilitates the high yield and purer CNTs compared
to Arc discharge method (Korneva 2008).

The
last method is CVD technique which has been extensively used in the synthesis
of SWCNTs recently. In this technique, Silica or Zeolite are commonly used as a
substrate while acetylene and cobalt are used as the catalyst and gas
respectively to produce SWCNTs (Edgington 2011). Since all the reactions are
conducted under low temperature, this technique is less expensive compared to
the Arc discharge and laser ablation method (Korneva 2008).

CNTs
have gained considerable interest as an anticancer drug delivery system due to
its unexpected thermal, mechanical and chemical properties. They are naturally
small in size compared to the most of drug delivery vesicles and able to
penetrate through the holes in tumours. Furthermore, they won’t be able to come
out from tumours after extravasation due to lack of lymphatic drainage (Raju et al. 2015). As discussed by Muguruma et al. (2007), CNTs are specially suitable for efficient drug delivery thanks to
their large surface to volume ratio as well as its hollow interior which can be
used to load hydrophobic drugs via non-covalent ?-? bonding ( Sanginario,
Miccoli & Demarchi 2017). Furthermore, chemically inert CNTs have
remarkable tensile strength and high elasticity in addition to their properties
like high electrical and thermal conductivity, ultra-lightweight, high
ductility and etc. (Micoli 2012).

However,
the toxicity of CNTs is considered as one of the main drawbacks and it
seriously undermines the usage of the CNT as a drug delivery system. The CNT’s
toxicity has been studied for decades, but still, its health and environmental
concerns are yet to be identified and specially those findings of CNTs are
always contradictory and far from adequate. For an instance, some studies
demonstrate that CNTs show some adverse effects such as oxidative stress,
apoptosis (Bottini et al. 2006; Manna
et al. 2005) whereas other studies
reported that no significant toxicity effects are associated with CNTs (Huczko
and Lange 2001; Pantarotto et al. 2004).

Many studies have been performed to figure
out the factors that cause the toxicity of carbon nanotube and it is found that
toxicity and reactivity of carbon nanotube are influenced by the several
factors such as metal contaminants, form of the CNTs, diameter, shape, length,
functionalization and etc (Lanone et al.
2013).

The
recent studies have demonstrated that catalyst metal contaminants which are
known as metal impurities is the most leading factor for toxicity of CNTs. The CNTs
can be contaminated by mixing with some catalysts residues such as Co, Fe, Ni,
and Mo during their preparation and purification process. The transition metals
can cause adverse effects on
health, for an instance peptide L-glutathione has antioxidant properties and
ability to protect cells from oxidative stress. However, its redox properties
are greatly undermined by the NiO metal impurities present in the SWCNT.

It is found that metal
residues present in the CNTs are shielded by graphitic shell. Therefore, metal
impurities in CNT can’t be completely removed without demolishing the
structural integrity of CNTs (Liu et al.
2007).

The
CNTs can present themselves in either form of fibres (CNTf) or form particles
(CNTp). It is found that CNTs might fit into the fibre category due to its
“fibre-like characteristics” (Donaldson et
al. 2012; Jaurand et al. 2009).
Therefore, studies demonstrated that their toxicology profile might also be
analogous to the other fibre particles like “asbestos fibres” (Jaurand et al.).
According to IRAC, asbestos exposure increases the risk of serious health
problems such as mesothelioma and lung cancer (National Cancer Institute 2017).
Thus, there is a very high probability that CNTs may have the same toxic
effects as asbestos fibres.

The shape of the CNTs is another key
factor that induces the toxicity. For an instance, a study has been done by using different shapes of CNT
including tangled CNT, long CNT, short CNT, carbon black crocidolite asbestos
and needle-like CNT to compare the effects of shape in inducing toxicity. The
results showed that pro-inflammatory responses are induced by long and
needle-like structure of CNTs in primary human macrophages (Palomaki et al.
2011). The Poland et
al. (2008) study has been done by introducing the SWCNTs into the abdominal cavity of the mice and the results showed
that CNTs induced the toxicity in the cavity by forming granules due to its
needle-like structure and it is further suggested that toxicity effects can be
minimized through the functionalization.

The degree of toxicity is varied by
different length of CNTs. Longer CNTs are more cytotoxic compared to shorter
CNTs and they tend to aggregate in cells since longer fibres cannot be engulfed
completely by macrophages (Liu et al.
2012, Yamashita et al. 2010). Sato et al. (2005) study was conducted to
investigate the inflammatory responses in human acute monocytic leukemia cell
line in vitro by using different lengths of MWCNTs which are 220 nm and 825 nm
and the results found that 825-CNT showed a high degree of inflammation
compared to 220 nm long CNT since 220-CNT can be readily enveloped by
macrophages compared to 825 nm long CNT.

 

The
pristine CNTs are poorly soluble in most of the biological media and they tend
to form toxic aggregates in the solution (Wang et al. 2016, Lawal et al. 2016). Therefore, considerable
works have been done by using different types of functionalized CNTs (f-CNT) to
reduce the cytotoxicity of CNTs and to increase their bioavailability. It has
been found that the toxicity of CNTs is greatly decreased by the
functionalization. Furthermore, low cytotoxic activities have been reported
from functionalized SWCNTs compared to functionalized MWCNTs (Azizian et al. 2010; Heister et al. 2009 &
Zhang. et al. 2009).

Functionalization
is the chemical modification which is achieved by either filling with some
particulate fluids inside the CNTs (endohedral) or introducing some specific
functional groups onto the surface of the CNTs (exohedral) (Micoli 2012). CNT functionalization can be divided
into two main categories which are endohedral and exohedral functionalization.
This proposal deals only with exohedral functionalization which can be further
classified into covalent and noncovalent functionalization based on the
mechanisms which are used to attach different functional groups and compound on
the sidewalls of the CNTs (Zhang et al.
2013).

Different
types of covalent reactions namely oxidation, 1,3-dipolar cycloaddition (Bianco, Kostarelos & Prato 2005) PEGylation (Razzazan et al.
2016) are used in covalent functionalization to graft molecules and
polymers on the sidewall sites or on the defects sites at the tips of the CNTs.
The strong and irreversible bonding between CNT and the attached molecule makes
the CNTs more stable (Micoli 2012). Two common covalent modifications namely oxidation
of the CNTs and PEG conjugation will be done in this study to compare the
toxicological profile of f-SWCNT with pristine CNT.

Oxidation
is one of the common approaches that widely employed for CNT modification. In
this method, oxidizing agents such as strong acids (nitric acid) are used to
generate the carboxylic groups at the reactive sites on CNTs (Bianco,
Kostarelos & Prato 2005). The oxygen-containing functionalities presence on
the graphite surface increase the solubility of the CNTs in the aqueous medium.
This modification is leading to debundling or exfoliation of the CNTs (Bose et al. 2010).

Polyethylene
glycol (PEG) is an amphiphilic polymer which readily dissolves in water and
many organic solvents. It is widely used with CNTs for conjugation purposes due
to its unique properties such as nontoxicity, high biocompatibility and
solubility (Razzazan et al. 2016). The PEGylated CNTs play a vital role in bio
distribution, it facilitates longer blood circulation time to maximize the
uptake by tumours and shows the Enhanced Permeability and Retention (EPR)
effects (Kushwaha et
al. 2013).

The
noncovalent functionalization is widely employed in CNT drug delivery
applications compare to the covalent functionalization. The conjugated
electronic structure of CNT is not perturbed by this functionalization, in
turn, electrical properties of the CNTs are also preserved (Rastogi et al. 2008). The noncovalent functionalization
is based on the weak forces such as ?- ? interactions, van der Waals and
hydrophobic interactions (Micoli 2012). Two common noncovalent modifications
namely PAA and Triton-X- 100 will be used in this study to compare the
toxicological profile of SWCNTs with pristine CNTs.

 

Chemical
moieties such as uncharged surfactants/ polymers can be linked to the CNT
graphite surface either via Coulomb attractions (Charged chemical moieties) or
?- ? stacking interactions. The main advantage of the surfactants, Triton-X-100
in particular, can be easily removed by washing. The Triton-X-100 has high
dispersing power compared to the most of surfactants such as SDS, Tween 20 and
Tween 80 (Rastogi et
al. 2008)

The goal of this study is to reduce the
toxicological profile associate with SWCNTs via using four different types of
functionalized single-wall carbon nanotubes (PEG-SWCNT, COOH-SWCNT,
Trion-X-100-SWCNT and PAA-SWCNT). Furthermore, this study deals with colorectal
cancer (CRC), since more than 6 million people die annually from this cancer
worldwide and in 2014, approximately 1.4 million new CRC cases and 50 000
deaths were reported in the USA (Patel 2014). Currently, not many studies have
been conducted to analyse the cytotoxic effects of f- SWCNTs in colorectal
cancers. Therefore, this study evaluates the toxicity responses of both
functionalized and pristine SWCNTs in in colon rectal cancer. Both covalent and
non-covalent functionalization of SWCNT will be elaborated in this study.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.0
Problem Statement

Colorectal cancer (CRC) is diagnosed as
the third most common cancer next to the lung and breast cancers (World cancer
research fund international 2016). CRC is conventionally treated with
chemotherapeutics agents which are associated with numerous side effects due to
their low target specificity. Therefore, there is a critical need to develop a versatile
delivery system which has ability distinguish affected cells from the normal
healthy cells (Patel 2014).

 

To date, a number of platforms have been
developed to treat colon cancer and among them, CNTs have gained considerable
interest as an anticancer drug delivery system, not only due to its unique
physiochemical properties but also the limitations associated with the current
drug delivery systems. For an instance, liposomes have low physical stability
and low entrapment efficiency compared to the CNTs. Furthermore, it is found
that liposomes might cause the “hand and foot syndrome” which is known as “palmar-plantar
erythrodysesthesia” due to its superficial toxicity and prolonged circulations
time (Mody et al. 2014).

 

The unique structural, electrical and
optical properties of the CNTs make them more attractive in drug delivery (Vashist et al.
2011; Siddiqui et
al. 2017). However, toxicity is one of the major drawback associated
with SWCNTs and very limited studies have been focused on their toxicity
effects. Furthermore, most of these findings are contradictory to each other. Therefore,
the toxicological profile of CNTs needs to be fully understood in order to use
them in clinical studies.

Most of the studied have been
demonstrated that toxic effects of CNTs can be reduced through
functionalization. Therefore, this study will use four different types of
functionalized SWCNTs to evaluate the cytotoxic profile of the CNTs.

 

 

 

 

 

 

 

 

 

3.0 Aim

 

3.1 General aim

In this study, it is proposed to evaluate in
vitro cytotoxic response of pristine SWCNTs and four different types of
functionalized SWCNTs in human colon cancer (WiDr) cells. 

 

3.2 Specific aims  

Followings are the specific aims of this
study

·        
To purify the pristine SWCNTs by using nitric
acid pre-sonication method

·        
To synthesise four different types of
functionalized SWCNTs (COOH-SWCNT, PEG-SWCNT, PAA-SWCNT and
(Triton-X-100-SWCNT) to represent the both covalent and noncovalent
functionalization

·        
To characterize the prepared SWCNTs

·        
To conduct the cytotoxicity
analysis of pristine and functionalized SWCNTs

 

 

4.0
Project Impact

 

SWCNTs have become one of the essential
platforms for active and passive cancer treatment due to its desired properties
which can cross the current limitations of most delivery systems (Kushwaha et al. 2013). The toxic profile of
SWCNTs can be minimized via functionalization. Therefore, the functionalized
SWCNTs (f-SWCNT) can be successfully used to deliver high load of the drug to
the target sites without having a risk of aggregation in the body. Furthermore,
health side effects associated with pristine CNTs can also be prevented from
using f-SWCNT.

 

 

 

 

 

 

 

 

 

5.0
Research Materials and Methodology

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1 –  The proposed methodology to evaluate the
cytotoxic responses of pristine and functionalized SWCNTs

 

 

5.1 Material

Pristine
SWCNTs will be purchased from Nanolab, Inc (length 1-5 ?m, purity > 80 %,
CVD method). WiDr human colon cancer cells will be used in this study as a cell
line. Furthermore, PEG, PAA and Triton -X-100 will be used to conduct the
functionalization of SWCNTs.

 

5.2 Methodology

5.2.1: Specific Aim 1

 To purify the pristine SWCNTs by using
nitric acid pre-sonication method

 

Pristine
SWCNTs are usually contaminated with amorphous carbon, metal catalyst particles
as well as graphitic nanoparticles. Therefore, SWCNTs will be purified by using
nitric acid pre-sonication method. In this method, the amount of 1 g of SWCNT
will be dispersed in aqueous HNO3 (2.6 M) and reflux it for 48 h.
The resulting solution will be centrifuged for 10 min and collect the sediment
for continuous washing with deionized water. After few cycles of washing with
deionized water, samples will be dried in vacuum for further usage.

 

5.2.2: Specific Aim 2

To
synthesise four different types of functionalized SWCNTs (COOH-SWCNT,
PEG-SWCNT, PAA-SWCNT and (Triton-X-100-SWCNT) to represent the both covalent
and noncovalent functionalization

 

Acid
Oxidation of SWCNTs

The concentrated acids will be used to
conjugate COOH to the sidewalls of SWCNTs. The volume of 45 mL of 98% H2SO4
and 15 mL 65% HNO3 will be added to purified SWCNTs. The resulting
mixture will be placed in an ultrasonic bath for 20 min at 40 °C prior to
stirring for 24 h while boiling under reflux. After the reaction mixture cools
down to the room temperature, it will be diluted with 1000 mL of deionized
water and use Millipore polycarbonate membrane (0.22 µm) to filter the
solution. This filtrate will be washed with 200 mL of THF and acetone to remove
the water from the filtrated solid. The carboxylated SWCNTs will be yielded
after drying above filtrated solid in a vacuum oven for 24 h at 50 °C.

 

 

 

PEG
conjugation SWCNTs (SWCNT-PEG)

PEGylation of SWCNT will be performed according to the method previously
reported in Razzazan et al. (2016)
study.

Preparation of PAA-SWCNTs

PAA-SWCNT complex will be prepared according to the method previously
reported in Liu et al. (2006). In
brief, it will be done by dispersing pristine SWCNT in 1 M of PAA under
sonication. The resulted dispersion will be undergone freeze-drying step to
obtain PAA-SWCNT.

Preparation
of (Triton X-100)- SWCNTs

The
(Triton X-100)-SWCNT will be prepared according to the protocol previously
reported in Rastogi et al. (2008). In
brief, Both Triton-X-100 and purified SWCNT will be ultrasonicated for   2 hours in order for surfactants to get
attach to the surface of SWCNT.

 

5.2.3: Specific Aim 3

To
characterize the prepared SWCNTs

 

The
chemical structure of the prepared samples will be analysed by Fourier
transform infrared spectroscopy (FTIR) and differential scanning calorimetry
(DSC). The morphological characterization will be done by scanning electron
microscopy. Transmission electron microscopy (TEM) will be further used to
obtain detailed information of the morphology on the prepared SWCNT.

 

5.2.4:  Specific Aim 4

To conduct cytotoxicity
analysis of pristine and functionalized SWCNTs

 

MTT assay

MTT assay will be conducted as described in Siddique et al. (2015) to determine the
percentage of cell viability. Briefly, the amount of 1 x 104 cells
will be seeded in 96-well cell culture plates and incubated for 24 h in CO2
incubator. After that 3-2, 5 -diphenyl tetrazolium bromide (MTT) will be added
to each well.

The plates will be incubated in CO2
incubator again for 4 h at 37 ?.  The volume of 200 µL of DMSO will be added to
each well and stirred gently after discarding the supernatant. Samples will be
analysed by UV spectrometer at 550 nm.

Neutral red uptake (NRU)
assay

NRU test will be carried out
according to the protocol described in Siddique et al. (2015) study. Plates will be analysed by UV-Spectrometer
under 550 nm and the obtained results will be compared with the control for
further analysis.

Morphological analysis by phase contrast microscope

Morphological analysis will be done to determine the
alternations in colon cancer line after expose to the pristine and
functionalized SWCNTs. The colon cancer cells (WiDr) will be tested with the volume of 10 µg/ml of f-SWCNTs
and pristine SWCNTs for 24 h and 48 h. Then inverted phase contrast microscope
(20 x) will be used to take the images of the cells.

Lipid peroxidation (LPO)

The test of lipid peroxidation will be conducted to
analyse the oxidative stress. Thiobarbituric acid-reactive substances (TBRAS)
will be used for LPO and this will be conducted as described in Dwivedi et al. (2014).

Reactive Oxygen species (ROS) generation

ROS generation will be done by using fluorescence agent
known as DCGH-DA according to the protocol described in Dwivedi et al. (2014) study. In brief, cells
will be incubated in 20 µM DCFH-DA
culture medium for 60 min in dark at room temperature after they wash with PBS.
Then fluorescence microscope will be used to analyse the intracellular
fluorescence of cells.

Mitochondrial membrane potential (MMP)

The effects of functionalized SWCNTs exposure on the MMP will be
analysed in WiDr cells. MMP test will be conducted according to the protocol
stated in Siddique et al. (2015). In brief, cells will be treated with Rhodamine-123
fluorescent dye and leave it for 1 h in dark at room temperature. Then
fluorescence microscope (x 20) will be used to measure the intensity of the
fluorescent dye.

Abstract

 

Single-wall carbon nanotubes have unique
physiochemical properties such as high drug loading efficiency, larger surface,
high thermal conductivity, which make them attractive in the range of
biomedical applications, particularly in anticancer drug delivery. However,
toxicity issues associated with CNTs have been raised. The cytotoxic profile of
the CNTs have not been fully understood yet and the available experimental data
related to CNT’s toxicity is also inconsistent. Hence, in this study, I will
evaluate the cytotoxicity effects of pristine and
functionalized single-wall carbon nanotubes (PEG-SWCNT, COOH-SWCNT,
Trion-X-100-SWCNT and PAA-SWCNT) on Widr colon cancer cells. Firstly, SWCNTs
will be purified and functionalized and then both pristine and functionalized
SWCNTs (f-SWCNT) will be characterized by using FTIR, DSC and TEM. The
cytotoxic responses of these f-SWCNT and pristine CNTs will be analysed using neutral
red uptake and MTT assays, Moreover, morphological analysis, mitochondrial
membrane potential, reactive oxygen species will be carried out in this study. The
hypothesis of this study is to observe the low toxicity effects from
functionalized CNTs compared to the pristine CNTs. This study will help to
understand the effects of functionalization on CNT toxicity. Most importantly,
functionalized SWCNTs can be successfully used as an anticancer drug delivery
system to deliver drugs to the target sites without having a risk of
aggregation in the body. Furthermore, health side effects associated with
pristine CNTs can also be prevented from using f-SWCNT.

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1.0 Introduction

 

Cancer is
responsible for the highest number of deaths in modern society next to the
heart and cerebrovascular diseases. According to WHO, cancer cause
approximately 8 million deaths annually all over the world. Most of the current
cancer treatments engage with the chemotherapeutic agents which have significant
toxicity because of its low target specificity (Kushwaha et al. 2013). As the result of the rapid
progress of nanotechnology, these conventional strategies started to replace
with some promising and innovative alternatives such as nanoscale drug delivery
systems. Among these platforms, carbon nanotubes (CNTs) have received a
significant attention in the last two decades due to its unique physiochemical
properties (Sanginario, Miccoli & Demarchi 2017).

CNTs
are cylindrical nanostructures which made of the sp2 hybridized
carbon atoms in a hexagonal arrangement. They can be categorized into two main
subgroups namely single-wall CNTs (SWCNTs) and multiwalled CNTs (MWCNTs)
depending on the number of graphite sheets that they constitute. Nanotube which
is formed by rolling up a single graphene sheet, is defined as SWCNT and those
which are formed by rapping more than one sheet in a concentric fashion are
known as MWCNTs (Kushwaha et al. 2013).
CNTs are capped at both ends of the carbon tube with a hemispherical
arrangement of a backyball structure. Furthermore, SWCNTs
have attracted considerable interest over the most of metal nanoparticles due
to their properties such as intrinsic stability, high encapsulation efficiency,
prolong circulation time, and etc (Singh et al. 2006; Wang
et al. 2004). Therefore, SWCNT
will be used in this study with an average diameter of 0.4-3 nm and an average
length of 1-5 ?m (Kushwaha et al.,
2013)

Several methods have been employed to
synthesize SWCNTs such as Arc discharge method, laser ablation method and
chemical vapour deposition (CVD) method. 
The Arc discharge method is one of the common technique which applies
the high DC current between two purity graphite electrodes to produce carbon deposit
at the cathode (Toma 2009). This method requires the further purification to
obtain pure and efficient SWCNTs (Edgington 2011).

Laser
ablation is the second most popular method to synthesis SWCNTs. In this method,
metal-graphite target is placed inside a tube, within a high-temperature
furnace and laser beam is scanned across the target surface to vaporize the
graphite target. Carbon nanostructures will be deposited outside of the furnace
on the water-cooled copper collector (Szabo et
al. 2010). This method facilitates the high yield and purer CNTs compared
to Arc discharge method (Korneva 2008).

The
last method is CVD technique which has been extensively used in the synthesis
of SWCNTs recently. In this technique, Silica or Zeolite are commonly used as a
substrate while acetylene and cobalt are used as the catalyst and gas
respectively to produce SWCNTs (Edgington 2011). Since all the reactions are
conducted under low temperature, this technique is less expensive compared to
the Arc discharge and laser ablation method (Korneva 2008).

CNTs
have gained considerable interest as an anticancer drug delivery system due to
its unexpected thermal, mechanical and chemical properties. They are naturally
small in size compared to the most of drug delivery vesicles and able to
penetrate through the holes in tumours. Furthermore, they won’t be able to come
out from tumours after extravasation due to lack of lymphatic drainage (Raju et al. 2015). As discussed by Muguruma et al. (2007), CNTs are specially suitable for efficient drug delivery thanks to
their large surface to volume ratio as well as its hollow interior which can be
used to load hydrophobic drugs via non-covalent ?-? bonding ( Sanginario,
Miccoli & Demarchi 2017). Furthermore, chemically inert CNTs have
remarkable tensile strength and high elasticity in addition to their properties
like high electrical and thermal conductivity, ultra-lightweight, high
ductility and etc. (Micoli 2012).

However,
the toxicity of CNTs is considered as one of the main drawbacks and it
seriously undermines the usage of the CNT as a drug delivery system. The CNT’s
toxicity has been studied for decades, but still, its health and environmental
concerns are yet to be identified and specially those findings of CNTs are
always contradictory and far from adequate. For an instance, some studies
demonstrate that CNTs show some adverse effects such as oxidative stress,
apoptosis (Bottini et al. 2006; Manna
et al. 2005) whereas other studies
reported that no significant toxicity effects are associated with CNTs (Huczko
and Lange 2001; Pantarotto et al. 2004).

Many studies have been performed to figure
out the factors that cause the toxicity of carbon nanotube and it is found that
toxicity and reactivity of carbon nanotube are influenced by the several
factors such as metal contaminants, form of the CNTs, diameter, shape, length,
functionalization and etc (Lanone et al.
2013).

The
recent studies have demonstrated that catalyst metal contaminants which are
known as metal impurities is the most leading factor for toxicity of CNTs. The CNTs
can be contaminated by mixing with some catalysts residues such as Co, Fe, Ni,
and Mo during their preparation and purification process. The transition metals
can cause adverse effects on
health, for an instance peptide L-glutathione has antioxidant properties and
ability to protect cells from oxidative stress. However, its redox properties
are greatly undermined by the NiO metal impurities present in the SWCNT.

It is found that metal
residues present in the CNTs are shielded by graphitic shell. Therefore, metal
impurities in CNT can’t be completely removed without demolishing the
structural integrity of CNTs (Liu et al.
2007).

The
CNTs can present themselves in either form of fibres (CNTf) or form particles
(CNTp). It is found that CNTs might fit into the fibre category due to its
“fibre-like characteristics” (Donaldson et
al. 2012; Jaurand et al. 2009).
Therefore, studies demonstrated that their toxicology profile might also be
analogous to the other fibre particles like “asbestos fibres” (Jaurand et al.).
According to IRAC, asbestos exposure increases the risk of serious health
problems such as mesothelioma and lung cancer (National Cancer Institute 2017).
Thus, there is a very high probability that CNTs may have the same toxic
effects as asbestos fibres.

The shape of the CNTs is another key
factor that induces the toxicity. For an instance, a study has been done by using different shapes of CNT
including tangled CNT, long CNT, short CNT, carbon black crocidolite asbestos
and needle-like CNT to compare the effects of shape in inducing toxicity. The
results showed that pro-inflammatory responses are induced by long and
needle-like structure of CNTs in primary human macrophages (Palomaki et al.
2011). The Poland et
al. (2008) study has been done by introducing the SWCNTs into the abdominal cavity of the mice and the results showed
that CNTs induced the toxicity in the cavity by forming granules due to its
needle-like structure and it is further suggested that toxicity effects can be
minimized through the functionalization.

The degree of toxicity is varied by
different length of CNTs. Longer CNTs are more cytotoxic compared to shorter
CNTs and they tend to aggregate in cells since longer fibres cannot be engulfed
completely by macrophages (Liu et al.
2012, Yamashita et al. 2010). Sato et al. (2005) study was conducted to
investigate the inflammatory responses in human acute monocytic leukemia cell
line in vitro by using different lengths of MWCNTs which are 220 nm and 825 nm
and the results found that 825-CNT showed a high degree of inflammation
compared to 220 nm long CNT since 220-CNT can be readily enveloped by
macrophages compared to 825 nm long CNT.

 

The
pristine CNTs are poorly soluble in most of the biological media and they tend
to form toxic aggregates in the solution (Wang et al. 2016, Lawal et al. 2016). Therefore, considerable
works have been done by using different types of functionalized CNTs (f-CNT) to
reduce the cytotoxicity of CNTs and to increase their bioavailability. It has
been found that the toxicity of CNTs is greatly decreased by the
functionalization. Furthermore, low cytotoxic activities have been reported
from functionalized SWCNTs compared to functionalized MWCNTs (Azizian et al. 2010; Heister et al. 2009 &
Zhang. et al. 2009).

Functionalization
is the chemical modification which is achieved by either filling with some
particulate fluids inside the CNTs (endohedral) or introducing some specific
functional groups onto the surface of the CNTs (exohedral) (Micoli 2012). CNT functionalization can be divided
into two main categories which are endohedral and exohedral functionalization.
This proposal deals only with exohedral functionalization which can be further
classified into covalent and noncovalent functionalization based on the
mechanisms which are used to attach different functional groups and compound on
the sidewalls of the CNTs (Zhang et al.
2013).

Different
types of covalent reactions namely oxidation, 1,3-dipolar cycloaddition (Bianco, Kostarelos & Prato 2005) PEGylation (Razzazan et al.
2016) are used in covalent functionalization to graft molecules and
polymers on the sidewall sites or on the defects sites at the tips of the CNTs.
The strong and irreversible bonding between CNT and the attached molecule makes
the CNTs more stable (Micoli 2012). Two common covalent modifications namely oxidation
of the CNTs and PEG conjugation will be done in this study to compare the
toxicological profile of f-SWCNT with pristine CNT.

Oxidation
is one of the common approaches that widely employed for CNT modification. In
this method, oxidizing agents such as strong acids (nitric acid) are used to
generate the carboxylic groups at the reactive sites on CNTs (Bianco,
Kostarelos & Prato 2005). The oxygen-containing functionalities presence on
the graphite surface increase the solubility of the CNTs in the aqueous medium.
This modification is leading to debundling or exfoliation of the CNTs (Bose et al. 2010).

Polyethylene
glycol (PEG) is an amphiphilic polymer which readily dissolves in water and
many organic solvents. It is widely used with CNTs for conjugation purposes due
to its unique properties such as nontoxicity, high biocompatibility and
solubility (Razzazan et al. 2016). The PEGylated CNTs play a vital role in bio
distribution, it facilitates longer blood circulation time to maximize the
uptake by tumours and shows the Enhanced Permeability and Retention (EPR)
effects (Kushwaha et
al. 2013).

The
noncovalent functionalization is widely employed in CNT drug delivery
applications compare to the covalent functionalization. The conjugated
electronic structure of CNT is not perturbed by this functionalization, in
turn, electrical properties of the CNTs are also preserved (Rastogi et al. 2008). The noncovalent functionalization
is based on the weak forces such as ?- ? interactions, van der Waals and
hydrophobic interactions (Micoli 2012). Two common noncovalent modifications
namely PAA and Triton-X- 100 will be used in this study to compare the
toxicological profile of SWCNTs with pristine CNTs.

 

Chemical
moieties such as uncharged surfactants/ polymers can be linked to the CNT
graphite surface either via Coulomb attractions (Charged chemical moieties) or
?- ? stacking interactions. The main advantage of the surfactants, Triton-X-100
in particular, can be easily removed by washing. The Triton-X-100 has high
dispersing power compared to the most of surfactants such as SDS, Tween 20 and
Tween 80 (Rastogi et
al. 2008)

The goal of this study is to reduce the
toxicological profile associate with SWCNTs via using four different types of
functionalized single-wall carbon nanotubes (PEG-SWCNT, COOH-SWCNT,
Trion-X-100-SWCNT and PAA-SWCNT). Furthermore, this study deals with colorectal
cancer (CRC), since more than 6 million people die annually from this cancer
worldwide and in 2014, approximately 1.4 million new CRC cases and 50 000
deaths were reported in the USA (Patel 2014). Currently, not many studies have
been conducted to analyse the cytotoxic effects of f- SWCNTs in colorectal
cancers. Therefore, this study evaluates the toxicity responses of both
functionalized and pristine SWCNTs in in colon rectal cancer. Both covalent and
non-covalent functionalization of SWCNT will be elaborated in this study.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.0
Problem Statement

Colorectal cancer (CRC) is diagnosed as
the third most common cancer next to the lung and breast cancers (World cancer
research fund international 2016). CRC is conventionally treated with
chemotherapeutics agents which are associated with numerous side effects due to
their low target specificity. Therefore, there is a critical need to develop a versatile
delivery system which has ability distinguish affected cells from the normal
healthy cells (Patel 2014).

 

To date, a number of platforms have been
developed to treat colon cancer and among them, CNTs have gained considerable
interest as an anticancer drug delivery system, not only due to its unique
physiochemical properties but also the limitations associated with the current
drug delivery systems. For an instance, liposomes have low physical stability
and low entrapment efficiency compared to the CNTs. Furthermore, it is found
that liposomes might cause the “hand and foot syndrome” which is known as “palmar-plantar
erythrodysesthesia” due to its superficial toxicity and prolonged circulations
time (Mody et al. 2014).

 

The unique structural, electrical and
optical properties of the CNTs make them more attractive in drug delivery (Vashist et al.
2011; Siddiqui et
al. 2017). However, toxicity is one of the major drawback associated
with SWCNTs and very limited studies have been focused on their toxicity
effects. Furthermore, most of these findings are contradictory to each other. Therefore,
the toxicological profile of CNTs needs to be fully understood in order to use
them in clinical studies.

Most of the studied have been
demonstrated that toxic effects of CNTs can be reduced through
functionalization. Therefore, this study will use four different types of
functionalized SWCNTs to evaluate the cytotoxic profile of the CNTs.

 

 

 

 

 

 

 

 

 

3.0 Aim

 

3.1 General aim

In this study, it is proposed to evaluate in
vitro cytotoxic response of pristine SWCNTs and four different types of
functionalized SWCNTs in human colon cancer (WiDr) cells. 

 

3.2 Specific aims  

Followings are the specific aims of this
study

·        
To purify the pristine SWCNTs by using nitric
acid pre-sonication method

·        
To synthesise four different types of
functionalized SWCNTs (COOH-SWCNT, PEG-SWCNT, PAA-SWCNT and
(Triton-X-100-SWCNT) to represent the both covalent and noncovalent
functionalization

·        
To characterize the prepared SWCNTs

·        
To conduct the cytotoxicity
analysis of pristine and functionalized SWCNTs

 

 

4.0
Project Impact

 

SWCNTs have become one of the essential
platforms for active and passive cancer treatment due to its desired properties
which can cross the current limitations of most delivery systems (Kushwaha et al. 2013). The toxic profile of
SWCNTs can be minimized via functionalization. Therefore, the functionalized
SWCNTs (f-SWCNT) can be successfully used to deliver high load of the drug to
the target sites without having a risk of aggregation in the body. Furthermore,
health side effects associated with pristine CNTs can also be prevented from
using f-SWCNT.

 

 

 

 

 

 

 

 

 

5.0
Research Materials and Methodology

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1 –  The proposed methodology to evaluate the
cytotoxic responses of pristine and functionalized SWCNTs

 

 

5.1 Material

Pristine
SWCNTs will be purchased from Nanolab, Inc (length 1-5 ?m, purity > 80 %,
CVD method). WiDr human colon cancer cells will be used in this study as a cell
line. Furthermore, PEG, PAA and Triton -X-100 will be used to conduct the
functionalization of SWCNTs.

 

5.2 Methodology

5.2.1: Specific Aim 1

 To purify the pristine SWCNTs by using
nitric acid pre-sonication method

 

Pristine
SWCNTs are usually contaminated with amorphous carbon, metal catalyst particles
as well as graphitic nanoparticles. Therefore, SWCNTs will be purified by using
nitric acid pre-sonication method. In this method, the amount of 1 g of SWCNT
will be dispersed in aqueous HNO3 (2.6 M) and reflux it for 48 h.
The resulting solution will be centrifuged for 10 min and collect the sediment
for continuous washing with deionized water. After few cycles of washing with
deionized water, samples will be dried in vacuum for further usage.

 

5.2.2: Specific Aim 2

To
synthesise four different types of functionalized SWCNTs (COOH-SWCNT,
PEG-SWCNT, PAA-SWCNT and (Triton-X-100-SWCNT) to represent the both covalent
and noncovalent functionalization

 

Acid
Oxidation of SWCNTs

The concentrated acids will be used to
conjugate COOH to the sidewalls of SWCNTs. The volume of 45 mL of 98% H2SO4
and 15 mL 65% HNO3 will be added to purified SWCNTs. The resulting
mixture will be placed in an ultrasonic bath for 20 min at 40 °C prior to
stirring for 24 h while boiling under reflux. After the reaction mixture cools
down to the room temperature, it will be diluted with 1000 mL of deionized
water and use Millipore polycarbonate membrane (0.22 µm) to filter the
solution. This filtrate will be washed with 200 mL of THF and acetone to remove
the water from the filtrated solid. The carboxylated SWCNTs will be yielded
after drying above filtrated solid in a vacuum oven for 24 h at 50 °C.

 

 

 

PEG
conjugation SWCNTs (SWCNT-PEG)

PEGylation of SWCNT will be performed according to the method previously
reported in Razzazan et al. (2016)
study.

Preparation of PAA-SWCNTs

PAA-SWCNT complex will be prepared according to the method previously
reported in Liu et al. (2006). In
brief, it will be done by dispersing pristine SWCNT in 1 M of PAA under
sonication. The resulted dispersion will be undergone freeze-drying step to
obtain PAA-SWCNT.

Preparation
of (Triton X-100)- SWCNTs

The
(Triton X-100)-SWCNT will be prepared according to the protocol previously
reported in Rastogi et al. (2008). In
brief, Both Triton-X-100 and purified SWCNT will be ultrasonicated for   2 hours in order for surfactants to get
attach to the surface of SWCNT.

 

5.2.3: Specific Aim 3

To
characterize the prepared SWCNTs

 

The
chemical structure of the prepared samples will be analysed by Fourier
transform infrared spectroscopy (FTIR) and differential scanning calorimetry
(DSC). The morphological characterization will be done by scanning electron
microscopy. Transmission electron microscopy (TEM) will be further used to
obtain detailed information of the morphology on the prepared SWCNT.

 

5.2.4:  Specific Aim 4

To conduct cytotoxicity
analysis of pristine and functionalized SWCNTs

 

MTT assay

MTT assay will be conducted as described in Siddique et al. (2015) to determine the
percentage of cell viability. Briefly, the amount of 1 x 104 cells
will be seeded in 96-well cell culture plates and incubated for 24 h in CO2
incubator. After that 3-2, 5 -diphenyl tetrazolium bromide (MTT) will be added
to each well.

The plates will be incubated in CO2
incubator again for 4 h at 37 ?.  The volume of 200 µL of DMSO will be added to
each well and stirred gently after discarding the supernatant. Samples will be
analysed by UV spectrometer at 550 nm.

Neutral red uptake (NRU)
assay

NRU test will be carried out
according to the protocol described in Siddique et al. (2015) study. Plates will be analysed by UV-Spectrometer
under 550 nm and the obtained results will be compared with the control for
further analysis.

Morphological analysis by phase contrast microscope

Morphological analysis will be done to determine the
alternations in colon cancer line after expose to the pristine and
functionalized SWCNTs. The colon cancer cells (WiDr) will be tested with the volume of 10 µg/ml of f-SWCNTs
and pristine SWCNTs for 24 h and 48 h. Then inverted phase contrast microscope
(20 x) will be used to take the images of the cells.

Lipid peroxidation (LPO)

The test of lipid peroxidation will be conducted to
analyse the oxidative stress. Thiobarbituric acid-reactive substances (TBRAS)
will be used for LPO and this will be conducted as described in Dwivedi et al. (2014).

Reactive Oxygen species (ROS) generation

ROS generation will be done by using fluorescence agent
known as DCGH-DA according to the protocol described in Dwivedi et al. (2014) study. In brief, cells
will be incubated in 20 µM DCFH-DA
culture medium for 60 min in dark at room temperature after they wash with PBS.
Then fluorescence microscope will be used to analyse the intracellular
fluorescence of cells.

Mitochondrial membrane potential (MMP)

The effects of functionalized SWCNTs exposure on the MMP will be
analysed in WiDr cells. MMP test will be conducted according to the protocol
stated in Siddique et al. (2015). In brief, cells will be treated with Rhodamine-123
fluorescent dye and leave it for 1 h in dark at room temperature. Then
fluorescence microscope (x 20) will be used to measure the intensity of the
fluorescent dye.

x

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