Synthesis of bioactive polyaniline- b -polyacrylic acid copolymer nanofibrils as an effective antibacterial and anticancer agent in cancer therapy, especially for HT29 treatment

Article abstract of DOI:10.1039/d0ra03779f

In this work, a new highly water-soluble copolymer of polyacrylic acid with polyaniline is introduced. Acrylic acid was polymerized via the Reversible Addition Fragmentation Chain Transfer method (RAFT) in the presence of an initiator and the obtained polyacrylic acid was copolymerized with aniline at room temperature. As the main achievements of this work, the resulting block copolymer with nanosized structure revealed favorable solubility in polar solvents, as well as excellent antibacterial and anticancer activities. Therefore, it is an appropriate candidate for medical applications such as wound healing and cancer therapy, especially in HT29 treatment.

Full text of DOI:10.1039/d0ra03779f

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PAPER
Synthesis of bioactive polyaniline-b-polyacrylic
acid copolymer nanofibrils as an effective
antibacterial and anticancer agent in cancer
therapy, especially for HT29 treatment
Cite this: RSC Adv., 2020, 10, 25290
a
Nazanin Bagheri,a Moslem Mansour Lakouraj,
Hamed Tashakkorian
Seyed Reza Nabavi,b
*
c
and Mojtaba Mohsenid
In this work, a new highly water-soluble copolymer of polyacrylic acid with polyaniline is introduced. Acrylic
acid was polymerized via the Reversible Addition Fragmentation Chain Transfer method (RAFT) in the
presence of an initiator and the obtained polyacrylic acid was copolymerized with aniline at room
temperature. As the main achievements of this work, the resulting block copolymer with nanosized
structure revealed favorable solubility in polar solvents, as well as excellent antibacterial and anticancer
activities. Therefore, it is an appropriate candidate for medical applications such as wound healing and
cancer therapy, especially in HT29 treatment.
Received 27th April 2020
Accepted 15th June 2020
DOI: 10.1039/d0ra03779f
rsc.li/rsc-advances
also improve the solubility of anticancer drugs.6 For example,
Takahashi et al. introduced new synthetic methacrylate random
1. Introduction
copolymers as anticancer agents by simulating the action of
anticancer drugs. In this research, the anticancer polymers were
designed to disrupt the cancer cell membranes. The polymers
exhibited cytotoxicity to proliferating prostate cancer, along
with killing (eliminating) dormant prostate cancer cells that
were resistant to docetaxel.7
Nowadays, there are many challenges for scientists to nd
efficient methods and materials in order to ght and minimize
potential health risks in the medical area. Many attempts have
been conducted toward preparing anticancer and anti-
microbial agents in the medical community. In this regard,
polymeric nanomaterials are the most widely used materials.
These types of nanoparticles provide a promising landscape for
researchers to improve the quality of treatment using modern
nanomedical methods. Based on the research, polymeric
treatments include polymer drugs,1 modied polymers with
protein and pharmaceuticals,2 and polymeric micelles.3 Due to
the variability in the methods of modication and macromo-
lecular synthesis, polymeric nanomaterials have found a wide
range of applications in treatment programs.
For improving the therapeutic performance of anticancer
drugs and the effectiveness of medication on cancer cells, the
anti-cancer agents should be controllably delivered to the
tumors.4 Polymeric materials compared with conventional
chemotherapy play an effective role in cancer therapies.5 In fact,
polymer nanomaterials not only reduce the toxicity of chemo-
therapy drugs to natural tissues around tumors, but they can
Undoubtedly, there is a close relationship between infection
and cancer. As a matter of fact, patients suffering from cancer
are susceptible to bacterial infections. Therefore, it is important
to investigate the medical care applicable to cancer patients in
terms of antibiotic effects. Several materials have been intro-
duced for antibacterial activity and are commonly applied as
antimicrobial agents that are non-volatile such as silver or
copper nanoparticles8 and quaternary ammonium compounds
(QACs).9 However, silver and copper, particularly in the form of
nanoparticles, have high cytotoxicity and low biocompati-
bility.10 In the case of QACs, some bacteria resist these mate-
rials, as well as cyto-compatibility with these quaternary
ammonium compounds.11 Hence, researchers have focused on
the modied biologically active polymers with more stability to
achieve higher performances in antibacterial properties. In fact,
the utilization of antibacterial non-vaporizable polymers leads
to the improved efficacy of the antimicrobial activity along with
diminished environmental risk.12,13
aPolymer Chemistry Laboratory, Department of Organic-Polymer Chemistry, Faculty of
Chemistry, University of Mazandaran, Babolsar 47416, Iran. E-mail: lakouraj@umz.
ac.ir
bDepartments of Applied Chemistry, University of Mazandaran, Babolsar 47416, Iran
cCellular and Molecular Biology Research Center (CMBRC), Health Research Institute,
Babol University of Medical Sciences, Babol, Iran
Among antimicrobial polymers, polycationic materials
provide effective antimicrobial activity due to the disruption of
negative charges on bacterial cell membranes, which lead to cell
death.14 In these antimicrobial polymers, cationic groups such
as ammonium, phosphonium and sulfonium ions interact with
dDepartments of Microbiology, Faculty of Basic Science, University of Mazandaran,
Babolsar 47416, Iran
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the bacterial cell membranes; simultaneously, the polymeric activity, making this synthetic copolymer a desirable candidate
residue may penetrate the microbial cell membranes, leading to to substitute for traditional chemotherapeutic drugs and also as
the distribution of cytoplasmic components and nally result- a strong antimicrobial agent in wound-dressing applications.
ing in the death of the bacterial cells.15–18 Consequently, cationic
polymers have stimulated signicant interest for application for
2. Materials
medical purposes,19 in the food industry,20 and in water sani-
tation21 to prevent the growth of bacteria.
Aniline (Merck Co.) was puried by double distillation under
Among the various types of antibacterial cationic polymers,
polyaniline is the most favorable bioactive polymer owing to its
good chemical stability and convenient synthesis.22 For
example, researchers have recently investigated the synthesis
and the antibacterial activity of the PANI–WO3 nano-
composites.23 The nanocomposites of PANI are more effective
against all the tested pathogenic strains. In addition, it was
proved that because of the greater surface area and higher
interactions of the polymer with the medium, nanostructured
polyaniline exhibits excellent dispersion in water, with low
toxicity to living systems.24 However, there are many disadvan-
tages regarding the use of these polymeric materials such as
decient solubility in most solvents due to the rigidity of the
conjugated benzenoid and quinoid rings, p–p stacking and
hydrogen bonding of polyaniline chains,25 which lead to low
processability of this compound. In this trend, researchers have
improved the solubility of polyaniline using different substitu-
ents on the polymer chains to prepare copolymers, or blending
polyaniline with other water-soluble polymers.26 For instance,
Shao et al. synthesized water-soluble polyaniline lms using
poly(2-acrylamido-2-methylpropane sulfonic acid) (PAMPS) as
a water-soluble dopant. Because of the hydrophilic nature of the
PAMPS, the nal blend lm was completely soluble in water.27
In another work, the synthesis of aqueous dispersions of PANI–
cellulose derivatives by polymerization of aniline monomer in
cellulose solution was reported. This cellulose derivative
revealed better conductivity and solubility, due to mutual
hydrogen bonding between the amine groups of polyaniline
chains and hydroxyl groups of cellulose. This phenomenon has
promoted the dispersion of PANI in the solution and prevents
its aggregation in the reaction medium.28
Considering the aforementioned points, and in order to
introduce a new water-soluble polymeric bioactive nano-
material, in this work, the nano-sized hydrophilic PANI copol-
ymer was synthesized by copolymerization of aniline with
polyacrylic acid. For this purpose, the aniline-functionalized
RAFT agent (CTA) was prepared and used to initiate the
controlled radical polymerization of acrylic acid in the presence
of AIBN to produce CTA-capped polyacrylic acid (CTA-PAA). The
sequential copolymerization of CTA-PAA with aniline was fol-
lowed through oxidative radical polymerization to afford the
corresponding block copolymer (PANI-b-PAA) in the nanometer
range. The prepared nanomaterials were characterized by
spectral data and physical properties. Thermal analysis was
conducted and the morphology, conductivity and biological
properties of the obtained copolymer were examined.
vacuum. All solvents, ammonium persulfate (APS), carbon
disulde (CS2), benzyl chloride, Na3PO4$12H2O, azobisisobu-
tyronitrile (AIBN), and hydrochloric acid (HCl) were purchased
from Merck and Sigma Companies and used without further
purication. Acrylic acid was supplied by Fluka Company. The
cell lines were purchased from the Pasteur Institute of Iran
(Department of Cell Bank).
3. Experimental
3.1. Synthesis of the chain transfer agent (CTA)
To a solution of aniline (0.016 mol) and hydrated sodium
phosphate (0.014 mol) (Na3PO4$12H2O) in 20 mL acetone, CS2
(0.06 mol) was added drop-wise and the mixture was allowed to
stir at room temperature for 2 hours. Benzyl chloride (0.02 mol)
was then added and the reaction mixture was kept stirring for 3
hours to give a light yellow precipitate. The resultant was
ltered and precipitated in cyclohexane. Finally, the product
was dried overnight at room temperature in a vacuum desic-
cator. The chemical structure was conrmed by IR and NMR
spectra that showed high purity.
ꢀ
Melting point: 68–70 C.
FTIR (KBr, n cmꢁ1): 1026, 1327, 1503, 1591, 2926 and
3118 cmꢁ1
.
1H-NMR (400 MHz, CDCl3): 4.58, 7.28, 7.30, 7.34, 7.38, 7.41,
7.43, 7.45 and 8.91 ppm.
3.2. Synthesis of CTA-capped polyacrylic acid (CTA-PAA)
The (CTA-PAA) macro-initiator was synthesized based on the
modied previously reported research.29 In a typical procedure,
acrylic monomer (5 mL) and CTA (0.12 g) were mixed in 25 mL
of THF. The mixture solution was degassed and stirred for 30
minutes at room temperature. Then, 0.014 g initiator (AIBN)
was added and the mixture was heated to 70 ꢀC and kept in
these conditions overnight. Aer completion of the reaction,
the resulting solution was poured into diethyl ether that led to
the formation of a sticky material. The solvent was decanted
and the product was dried in a vacuum oven at 60 ꢀC for 12
hours. The obtained white powder was characterized by IR and
NMR spectroscopies.
FTIR (KBr, cmꢁ1): 1248, 1454, 1709, 2932 and 3436 cmꢁ1
.
1H-NMR (400 MHz, DMSO-d6): 1.76, 2.50, 3.60, 5.88, 6.07,
6.22, 12.25 ppm.
3.3. Synthesis of polyacrylic acid-b-polyaniline copolymer
(PANI-b-PAA)
The main achievement of this work is that the resulting
polymeric nanomaterial exhibited extra solubility in polar The synthesis of the PANI-b-PAA copolymer was carried out
solvents such as ethanol, H2O and DMSO, and surprisingly according to the following procedure: to a mixture of CTA-PAA
indicated excellent inherent antibacterial and anticancer (1 g) and 30 mL HCl (1 M), ammonium persulfate (0.001 mol)
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was added and stirred for 30 minutes at room temperature.
Then, aniline (0.01 mol) was added and the mixture was stirred
for over 4 hours till the color changed to dark green. Aer that,
the resultant was washed with large quantities of ethanol and
distilled water (50 : 50 v/v) for 2 hours and then it was centri-
fuged to separate the insoluble homopolymer of aniline. The
nal supernatant was concentrated to give the PANI-b-PAA
copolymer as a green precipitate. The chemical structure of the
copolymer was identied by FTIR and NMR spectroscopy.
FTIR (KBr, cmꢁ1): 1135, 1483, 1556, 1648, 1710, 2920 and
5.1. Fourier transform infrared spectroscopy (FTIR)
FTIR spectra of CTA, CTA-PAA and the block copolymer (PANI-b-
PAA) are shown in Fig. 1. The spectrum of CTA (Fig. 1a) showed
two sharp bands about 1026 and 1326 cmꢁ1, which are attrib-
uted to the stretching vibrations of C–S and C]S bonds,
respectively. Also, peaks at around 1503 and 3118 cmꢁ1 indi-
cated vibrations of C]N and N–H.
As can be seen in Fig. 1b, CTA-PAA displayed peaks at around
1248, 1710 cmꢁ1 and a broad band from 2800 to 3500 cmꢁ1
,
which clearly demonstrate the characteristic stretching vibra-
tions of C–O, C]O (carboxylic acid) and acidic O–H, respec-
tively. In the case of the PANI-b-PAA copolymer, the spectrum
(Fig. 1c) exhibited corresponding stretching vibrations of C]O,
aliphatic C–H and O–H bonds at around 1710, 2920 and
3436 cmꢁ1, respectively. Also, the formation of the PANI
segment in the copolymer was conrmed by (or through) the
clear peaks at 1556 cmꢁ1, corresponding to the C]C stretching
of the quinoid ring, and 1483 cmꢁ1 for the C]C stretching
vibration of the benzenoid ring, as well as 1297 cmꢁ1 related to
C–N stretching.
3436 cmꢁ1
.
1H-NMR (400 MHz, DMSO-d6): 2.50, 4.02, 7.14, 7.16, 7.19,
7.34, 7.45, 10.06 ppm.
4. Characterization
The Fourier transform infrared (FTIR) spectra of all samples
were recorded at 400 to 4000 cmꢁ1 from KBr pellets on a Bruker
1
infrared Fourier transform spectrometer. The H-NMR spectra
of all compounds were accomplished using a Bruker 400 MHz
Ultra-shield Advance DRX spectrometer in dimethyl sulfoxide
(DMSO-d6) and CDCl3 as solvents. The X-ray powder diffraction
patterns were recorded on a PHILIPS (PW1730) diffractometer
tted with CuK a radiation (l ¼ 1.5404 nm) at 40 kV and 30 mA
in the 2q range of 5–60ꢀ. Thermal analyses (DSC and TGA) were
performed using a TA (Q600) instrument operating at a heating
rate of 20 C min , from room temperature up to 350 C for
TGA and 600 ꢀC for DSC under a N2 atmosphere. Melting points
of low molecular weight organic compounds were measured
using the Electrothermal apparatus. Scanning electron
microscopy (SEM) images of samples were taken by KYKY-
EM3200. Moreover, the standard four-point probe (Azar Elec-
trode Company, Tabriz, Iran) method was applied to measure
the conductivity at room temperature. The electrospinning of
the nal copolymer was investigated and eventually, biological
tests such as antibacterial and cytotoxicity assays (MTT test)
were examined for the copolymer.
5.2. NMR spectroscopy
1H-NMR spectrum of the synthesized CTA represents the char-
acteristic peaks at corresponding chemical shis, i.e., 4.58 ppm
related to aliphatic CH2, 7.28 to 7.45 ppm for aromatic hydro-
gens and 8.91 ppm attributed to NH. Furthermore, for the CTA-
PAA sample, the chemical shis in the range of 1.50 to 2.50 ppm
are related to the aliphatic CH and CH2 units of the polyacrylic
acid chain. Additionally, the peaks observed from 5.90 to
6.29 ppm pertain to the aniline ring, and the characteristic peak
at 12.25 ppm is ascribed to the acidic protons of carboxylic acid
groups. Finally, for the synthesized PANI-b-PAA copolymer, the
chemical shi of 4.01 ppm is related to aliphatic CH, and the
triplet peaks located at 7.16, 7.29 and 7.42 ppm are associated
with polyaniline. Moreover, the chemical shi at 10.06 ppm
refers to acidic protons in the acrylic acid units. These data
clearly indicate the successful RAFT copolymerization of aniline
and acrylic acid (Fig. 2). Also, the molecular weight of the
polymers was calculated based on 1HNMR ndings as reported
in previous research37,38 using the integration of peaks from
Fig. 2B and C, which are equivalent to 13 180 and 9950 kDa for
polyacrylic acid and polyaniline, respectively.
ꢁ1
ꢀ
ꢀ
5. Results and discussion
Over the last few decades, living radical polymerization has
been utilized as one of the most striking and applicable
methods for the synthesis of diverse novel polymers. Among the
various techniques for radical polymerization,30,31 the Revers-
ible Addition Fragmentation Chain Transfer (RAFT) is the most
attractive one in living or controllable radical polymeriza-
tions.32–34 This kind of controlled radical polymerization is
associated with thiocarbonylthio compounds as chain transfer
agents and, in particular, has been applied for the preparation
of various structures such as block, gra, hyperbranched and
star copolymer with ideal dispersity and narrow molar mass
5.3. Conductivity measurements
The electrical conductivity of the copolymer (PANI-b-PAA) was
studied by a four-probe technique. In this way, the powdered
copolymer (0.3 g) was prepared in a tablet form (diameter: 13
mm, thickness: 1 mm) at a pressure of 14 MPa. The conductivity
was measured by the following equation:
ln 2
I
s ¼
ꢂ
pd
V
distribution.35,36 Therefore, the RAFT polymerization method where s is the electrical conductivity, V is the voltage measured
was applied for the synthesis of polyacrylic acid-b-polyaniline to across inner probes, (I) is the applied current in the outer
produce an amphiphilic bioactive water-soluble polymeric probes, and (d) is the tablet thickness (cm). The electrical
system (Scheme 1).
conductivity of the copolymer was 18 S cmꢁ1, whereas the
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Scheme 1 Synthetic pathway for the polyacrylic acid-b-polyaniline copolymer.
Fig. 1 FTIR spectra of CTA (a), CTA-PAA (b) and PANI-b-PAA (c).
electrical conductivity of the polyaniline homopolymer re- cooperate in the charge transfer through the internal doping
ported in the literature was lower than in the present work.39,40 of carboxylic acid. Also, it was assumed that owing to the
It seems that the increase in the electrical conductivity of the existence of the polyacrylic acid chain, the solubility of the
copolymer is due to the presence of the polyacrylic acid block copolymer was improved, which led to an increment in the
in the PANI-b-PAA copolymer, which acts as an internal amount of aniline units in the polyaniline segments during
dopant. It was suggested that carboxylate groups might polymerization.
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Fig. 2 1H-NMR spectra of CTA (A), CTA-PAA (B) and PANI-b-PAA (C).
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Fig. 3 SEM images of PANI-b-PAA.
is expected that with the increase in the pore numbers and the
volume, the cell adhesion and proliferation will be
enhanced.41 SEM images proved that the electrospinning
process of PANI-b-PAA was successful for the presentation of
electrically conducting domains at the surface, which may be
effective for transferring electrical signals (Fig. 4).
5.4. Scanning electron microscopy (SEM)
The nanostructure of the synthesized PANI-b-PAA copolymer
and the size of the nanoparticles were evaluated by SEM
imaging (Fig. 3). SEM images demonstrated that the PANI-b-PAA
copolymer has a nano-rod shape with a diameter range of 52 to
100 nm.
5.6. XRD analysis
5.5. Electrospinning of the copolymer
The X-ray diffraction pattern of CTA-PAA and PANI-b-PAA
copolymers are given in Fig. 5. The XRD pattern of PANI-b-PAA
(the violet curve) contains some sharp peaks representing the
crystal planes. The peak centered at 2q of about 21.20ꢀ may be
attributed to the conjugated chain of the emeraldine base form
of PANI.42
Electrospinning is a method in which materials in solution or
melt states are transformed into nano- or micro-sized
continuous bers. To study the presumption of electro-
spinning of the copolymer, the resulting PANI-b-PAA solution
was loaded into two 10 mL glass syringes equipped with
a needle. The syringe was then put in a syringe pump and the
needle was connected to the positive output of a high voltage
power supply. The collector was covered with aluminum foil
and placed at a xed distance of 20 cm from the needle. The
ow rate of the solution and applied voltage were xed at 1 mL
per hour and 18–20 kV at room temperature, respectively.
Finally, the prepared PANI-b-PAA nanobers on the foil were
As shown in Fig. 5, the red curve, the CTA-RAFT showed
a
moderate degree of crystallinity (broad peaks in the
spectrum).
5.7. Thermal analysis
5.7.1. Thermogravimetric analysis (TGA). Thermal analysis
dried overnight at room temperature. The morphology of the of the CTA-PAA and PANI-b-PAA were performed under a N2
ꢀ
nano-brous scaffolds was examined using scanning electron atmosphere from room temperature to 550 C [Fig. 6a and b].
microscopy. To estimate the diameter, the images were The TG-DTG curve for CTA-PAA showed several stages. The rst
captured, which indicated that the diameters of the bers stage of weight loss at around 100 ꢀC represents the removal of
were around 80 nm.
H2O and other volatile components. In the next stage, the
SEM images show the morphology and distribution of the dithiocarbamate functional group underwent decomposition at
prepared nanobers. As is evident, the non-woven bers were ca. 150 ꢀC. Besides, the weight loss at around 250–300 ꢀC arises
distributed by chance throughout the electrospun structures from the decarboxylation of carboxylic acid on PAA segments,
and formed interconnected pores. As mentioned elsewhere, it and the nal stage (400 ꢀC) represents the oxidative degradation
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Fig. 4 SEM images and the distribution of the prepared nanofibers of electrospun PANI-b-PAA.
of carbon chains.43 As presented in the TGA curve for PANI-b- the end-capped polyacrylic acid chain. Elimination of carboxyl
ꢀ
PAA (Fig. 6b), the rst weight loss occurred at around 100 C, groups was observed at 200–300 ꢀC and eventually, the main
ꢀ
resulting from the removal of entrapped water. The second weight loss at around 450–500 C originated from the polyani-
stage (200 ꢀC) refers to the loss of dithiocarbamate groups in the line chain degradation.
PANI-b-PAA copolymer, which revealed the increase in the
5.7.2. Differential scanning calorimetry (DSC). DSC ther-
thermal stability of the dithiocarbamate linkage in the copol- mograms of CTA-PAA and PANI-b-PAA are given in Fig. 7a and b.
ymer along with the formation of the polyaniline segment on The DSC thermogram of CTA-PAA shows ve endothermic
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Fig. 5 The XRD patterns of CTA-PAA (red curve) and PANI-b-PAA (violet curve).
peaks and one exothermic peak. The rst peak was attributed to cultures of the test organisms, which were spread as regularly
ꢀ
the removal of water at 50–100 C and the second one can be as possible throughout the entire media. The tablets were
related to the melt-crystal domains of the polyacrylic acid at presented on the upper layer of the seeded agar plates and
131.5 ꢀC. Peaks at 175 ꢀC and 186 ꢀC can be ascribed to the were incubated at 37 ꢀC for 24 h. The antibacterial activity of
degradation of thioester groups in dithiocarbamate linkages. the copolymer was compared to the famous antibiotics
The exothermic peak at 209 ꢀC is attributed to the crystallization gentamicin (10 mg per disk) and chloramphenicol (30 mg per
of PAA. Due to the broad submerged peaks, it was not possible disk) as positive controls. The antibacterial activity was
to clearly detect the Tg point. In Fig. 7b, the DSC curve of PANI-b- estimated by measuring the diameter of the inhibition zone
PAA unveils two endothermic peaks and one exothermic peak. (mm) on the surfaces of the plates and the results are re-
The rst peak corresponds to the removal of water at 50–100 ꢀC ported as the mean
and the melt-crystal domains of the polyacrylic acid. The second measurements.
ꢃ
SD values aer two repeated
peak accounts for the decarboxylation in the polyacrylic acid
The results are summarized in Table 1. As can be seen, the
chain and the exothermic peak at 315 ꢀC may be assigned to the antibacterial activity evaluation of all the copolymeric tablets
crystallization of PAA or the cross-linking/oxidation of the PANI- was performed on four different bacterial strains (Gram-
b-PAA.
positive: Staphylococcus aureus (S. aureus) and Bacillus subtilis
(B. subtilis) and Gram-negative: Escherichia coli (E. coli) and
Pseudomonas aeruginosa (P. aeruginosa)) using an agar disc
diffusion technique [Fig. 8].
5.8. Determination of antibacterial activity
The antibacterial activity of the synthesized materials was
assessed by a Kirby–Bauer disk diffusion method where a disk
was placed on the surface of agar dishes.44 The PANI-b-PAA (0.13
g) was prepared in tablet form (diameter: 13 mm, thickness: 1
mm) at a pressure of 14 MPa. Next, 8 prepared tablets were
placed on agar dishes infected with both Gram-negative and
Gram-positive species of bacteria. The antibacterial activity of
the synthesized copolymer was studied against four bacterial
species (Escherichia coli PTCC 1330, Pseudomonas aeruginosa
PTCC 1074, Staphylococcus aureus ATCC 35923 and Bacillus
subtilis PTCC 1023). All the microorganisms were protected
The results showed that the antibacterial activities of the
PANI-b-PAA block are obviously stronger than standard antibi-
otics. Furthermore, by comparing these results with previous
reports, it was found that the present synthesized copolymer
disclosed reasonable antibacterial activity [Table 2].
As shown in Fig. 9, it was suggested that the antimicrobial
mechanism of the copolymer may be due to the presence of
nano-sized amphiphilic polyaniline brils in the copolymer
structure. It was assumed that polyaniline segments with
positive charges disrupted the bacterial cell membranes,
causing leakage of the cytoplasmic contents or complete lysis of
the cells and consequently the death of the bacteria as was re-
ported previously in the literature.48
ꢀ
(37 C, 24 h) by inoculation in a Mueller–Hinton broth.
The cultures were standardized with a nal cell density of
around 108 cfu mLꢁ1. The Mueller–Hinton agar (Merck)
samples were prepared and inoculated from the standardized
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Fig. 6 TGA-DTG curves of CTA-PAA (a) and PANI-b-PAA (b).
fresh culture medium for 2 to 5 passages. Primary human
broblasts were isolated from fresh foreskin extracted from
children aged between 1.5 and 30 months who underwent
routine circumcision as previously described.49 Briey, 1 to 2
mm3 pieces of foreskin were incubated at 37 ꢀC for 2 hours in
a 15 mL falcon tube in the presence of 0.5% dispose II (Sig-
ma-Aldrich) and were then digested with 0.1% crude colla-
genase (Sigma-Aldrich, C2674). Cells were collected upon
release every 20 minutes from the aqueous phase of the tube,
passed through a 70 mm cell strainer, and centrifuged before
resuspending in high glucose Dulbecco's modied Eagle's
medium supplemented with 10% FBS and 1% penicillin/
streptomycin. Written informed consent letters were ob-
tained from the parents of all the children who received
a routine circumcision at Amirkola Children's Hospital
(Babol, Iran), and the study was approved by the Ethical
Committee of Babol University of Medical Sciences
(Mubabol.rec.1389.3).
5.9. Cytotoxicity assay (MTT test)
5.9.1. Sample preparation. The synthesized PANI-b-PAA
copolymer was dissolved in DMSO, giving a stock concentration
of 20 mmol dmꢁ3. Aerwards, 62.5, 125, 250, and 500 mg mLꢁ1
solutions were obtained by diluting in the appropriate medium
supplemented with 10% (mg mLꢁ1) fetal bovine serum (FBS) and
1% mixture of penicillin/streptomycin. All prepared samples
were sterilized under standard conditions using an autoclave
for further experiments.
5.9.2. Cell culture. The HT29 (human colon cancer cell
line) and PC3 (prostate cancer cell line) were obtained from
the National Cell Bank of Iran (Pasteur Institute of Iran). The
cell lines were cultured in RPMI-1640 (Sigma-Aldrich, Ger-
many) and fortied with 10% FBS (Gibco/Invitrogen) and 1%
penicillin and streptomycin (Sigma-Aldrich, USA) in 5% CO2
at 37 ꢀC with 95% air humidity. The culture medium was
changed every 2 days aer washing with phosphate-buffered
saline (PBS). This procedure was performed with 10 mL of
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Fig. 7 DSC curves of CTA-PAA (a) and PANI-b-PAA (b).
solution was added to the wells. Aer 4 hours, the previous
MTT solution was replaced by 200 mL of MTT solvent (acidic
isopropanol) for the solubilization of formazan crystals. The
mean absorbance of the plate was measured using an Elisa
reader at 570 nm.
5.9.3. MTT assay. The HT29 and PC3 cell lines were
cultured in well plates; 200 mL of the culture medium con-
taining 10% (mg mLꢁ1) FBS was added to each well. Aer 48
hours under standard conditions, the culture medium was
removed, and the cultured cells were treated with fresh culture
medium with different concentrations of the polymeric
sample as described previously. The cultured cells were kept
in an incubator for 48 and 72 hours. Then, 200 mL of the
5.10. Anti-cancer activity
culture medium was removed, and 50 mL of MTT (5 mg mLꢁ1
)
Colorectal cancer is the third most common cancer in the world
that causes cancer death every year.50 As reported in previous
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Table 1 Antibacterial activity of the PANI-b-PAA film using the Kirby– Table
2
Comparison of inhibition zone values of PANI-based
Bauer technique (zone of growth inhibition, mm)
materials
Zone of growth inhibitiona (mm)
Zone of growth inhibition (mm)
Gentamicin
Chloramphenicol
S. aureus
(positive type)
Test strain
PANI-b-PAA (lmb) (10 mg per disc) (30 mg per disc)
Test strain
E. coli (negative type)
E. coli
28.0 ꢃ 1.4
19.6 ꢃ 1.1
15.6 ꢃ 0.5
20.3 ꢃ 1.5
26.0 ꢃ 1.7
20.7 ꢃ 1.5
NEc
Pure PANI45
10 ꢃ 0.4
13 ꢃ 0.4
33.3
—
12
11 ꢃ 0.5
16 ꢃ 0.4
35.9
—
15
P. aeruginosa 28.5 ꢃ 0.7
PANI–cellulose45
PANI–Cu0.05Zn0.95O46
PANI–PVA blend47
PANI–PVA–Ag (15%)47
Present work
S. aureus
B. subtilis
31.5 ꢃ 0.7
27.5 ꢃ 0.7
21.7 ꢃ 0.6
22.3 ꢃ 1.2
a
Strong activity > 16 mm, moderate activity 10–16 mm, weak activity <
28.0 ꢃ 1.4
31.5 ꢃ 0.7
b
c
10 mm. Diameter of lm: 10 mm, Mueller–Hinton agar plate. No
effect.
hematological reactions because of their undened distribution
in the bone marrow, liver and other living tissues.53 Moreover,
most drugs that have been applied in the treatment of colorectal
cancer exhibit many side effects of chemotherapy such as hair
loss, mouth sores, loss of appetite, vomiting, increased chance
of infections and fatigue. Thus, scientists have attempted to
develop novel nanomaterial systems with multifunctional
characteristics that control the release of drugs.
works, the common drugs available for colorectal cancer (CRC)
treatment include 5-uorouracil (5-FU)/Leucovorin, the rst-
line treatment, Avastin, Oxaliplatin and Irinotecan.51 Fluo-
ropyrimidines, Oxaliplatin and Irinotecan have been made
public as chemotherapy drugs for CRC. Unfortunately, one of
the most serious problems of chemotherapy treatment for
colorectal cancer involves using high doses of medicines,
leading to the cytotoxicity of healthy cells, especially in the case
of the mixture of 5-FU and Irinotecan.52 Also, it is noteworthy
that these traditional medicines always cause severe
For this purpose, to determine the cytotoxic effect of the
synthesized PANI-b-PAA sample, the treatments of two kinds of
Fig. 8 Photograph of the antibacterial activity assays of the PANI-b-PAA.
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Fig. 9 Leakage of the cytoplasmic contents of the cells by the copolymer.
cancer cell lines, HT29 and PC3 cell lines and broblasts were decrement but in the case of HT29, an impressive decrease in
investigated and the results are demonstrated in Fig. 10. cell proliferation was observed aer 72 h. This means that,
The outcomes attained from Fig. 11 revealed that cell fortunately, the synthesized PANI-b-PAA copolymer in
proliferation was decreased in the broblast cell lines from a maximum concentration of 125 mg mLꢁ1 shows effective
concentrations of 250 mg mLꢁ1 to 500 mg mLꢁ1. Moreover, as activity for the reduction of human colon cancer cell growth.
shown in Fig. 11, by increasing the copolymeric sample Therefore, the PANI-b-PAA copolymer is
a satisfactory
concentration, PC3 cell lines did not show any phenomenal
Fig. 10 Proliferation decrement of fibroblasts, colonic cancer cells (HT29), and prostate cancer cells (PC3).
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Fig. 11 Fibroblast (A), HT29 (B) and PC3 (C) cell proliferation under treatment with different concentrations of PANI-b-PAA samples in
concentrations of 62.5, 125, 250 and 500 mg mLꢁ1 in 48 and 72 hours.
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candidate for human colon cancer treatment as an effective,
simple preparation and cheap treatment protocol.
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In the present work, the novel water-miscible conducting
diblock copolymer (PANI-b-PAA) was successfully synthesized by
sequential RAFT and oxidative radical polymerizations. The
obtained products were characterized by different analyses. The
nanocrystalline structure of PANI-b-PAA was proved by XRD and
SEM imaging. The nal sample, PANI-b-PAA, showed reason-
able solubility in polar solvents such as H2O, ethanol, a mixture
of H2O and ethanol and DMSO. Also, further investigations
revealed excellent antibacterial activity for the PANI-b-PAA
copolymer on four bacteria, namely, E. coli, P. aeruginosa, B.
subtilis, and S. aureus. Moreover, the anti-cancer activity of the
synthesized copolymer was investigated. Regarding the MTT
assay, the outcomes showed promising results in decreasing the
cancer cell proliferation of HT29 at a concentration of 125 mg
mLꢁ1. The combination of the antibacterial properties with
anticancer features makes the PANI-b-PAA copolymer a desir-
able polymeric nanomaterial for cancer treatment. In chemo-
therapy, since the infection may occur in and around cancerous
organs, antibacterial activity makes the introduced material
more operative for cancer treatment. All the results demonstrate
that this charged amphiphilic block copolymer is a proper
candidate for biological applications such as the anticancer
agent in colorectal cancer. Furthermore, owing to excellent
antibacterial properties, it possesses potential activity for
wound dressing applications. Therefore, we are hoping that by
employing this synthesized copolymer we will be able to reduce
the side effects of traditional anti-cancer medicines without
using any additional drugs.
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Conflicts of interest
There are no conlict of interest in any form to declare.
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Acknowledgements
We appreciate the partial nancial support of the research
Council of the University of Mazandaran.
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