This review discusses light-activated conjugated polymers and their potential use in combating cancer. Certain water-soluble conjugated polymers like polythiophene are effective against tumor cells as therapeutic agents and optical imaging markers. They produce reactive oxygen species that promote apoptosis in tumor cells through oxidative stress when exposed to fluorescent light. Frequency resonance energy transfers are used to more efficiently produce reactive oxygen species from the polymers. Polymers like polythiophene and its derivative PTPF show selective anticancer activity against specific tumor cells and low toxicity towards healthy tissue. They can be used to optically image cancer treatment and distinguish between living and dead cells.

1. Chemical Society Reviews RSCPublishing
Tutorial Review
This journal is © The Royal Society ofChemistry 2013 Chem. Soc. Rev., 2014, 01, 1-5 | 1
Received 24th March 2014,
Accepted 24th
March 2014
Light Activated Conjugated Polymers with
Anticancer Activity
David A. McMillana
The purpose of this review is to describe and outline the mechanisms and relevancy of light
activated conjugate polymers in the biomedical world. Certain water soluble conjugat ed
polymers like polythiophene (PTP) are widely used as therapeutic molecules against tumor
cells as well as optical imaging markers. Frequency resonance energy transfers (FRET) are
used to produce reactive oxygen species (1
O2) to further promote apoptosis in tumor cells by
method of oxidant stress. The fluorescent properties of these polymers help research er s
distinguish between living and dead cells throughout the process by using optical microscop y .
It will be shown, that conjugated polymers are not only effective in combating cancer cells
when exposed to fluorescent light; they also wield very low cytotoxicity levels which do not
harm surrounding healthy tissue. Also, with the develop ment of PTP, scientists were able to
create water soluble polymers (such as PTPF) which demonstrate selective anticancer activity
towards specific tumor cells.
1. Introduction
When an individual is diagnosed with an illness such as
cancer, there are many different treatment paths one can take.
One treatment plan mentioned regularly by physicians is
chemotherapy. Chemotherapy involves the injection of certain
drugs into the patient’s body to prevent cancerous cells from
advancing at certain stages of the illness. Essentially, the drugs
are meant to stop tumor cells from spreading by promoting
apoptosis.1
Conjugated polymers are becoming more involved in a
variety of industrial applications worldwide. It has grown into a
multi-million dollar industry where most of the polymers
developed are replacing traditional standard polymers that are
already widely accepted. One field where it is gaining a lot of
attention and popularity is biomedical imaging and therapeutics.
Using the technique of fluorescence imaging, researchers are
able to understand the mechanisms and functions of biological
systems, such as cancer cell growth. By using optically active
polymers with this technique, researchers can monitor the
progress of a conjugated polymer as it kills cancerous cells in a
tissue mass.2
Conjugated polymers are the more preferred subject
to use for cancer tumors because of their low cytotoxicity ,
sensitivity to certain cancer types, and imaging ability using
fluorescent light. Their structure also contains a backbone that
consists of delocalizing and semiconducting characteristics. In
comparison to other molecules, CP’s can transfer the excited
energy from their backbone to lower energy electron/ener gy
acceptor sites over long distances. This enables them to wield an
intense fluorescent signal once initially excited by an external
source.3
Solubility of polymers in biomedical imaging is important
because the molecules they interact with are enveloped in
aqueous media. The basic structure of water soluble conjugated
polymers (WSCP’s) contains two sections: the first sections are
a π-conjugated backbone that gives them their optical properties
that determine absorption and emission spectra. The other
sections are their charged functional groups which make them
soluble in water.4
Over the past few decades, WSCP’s are
becoming a standard platform for optical and sensitive imagin g
in biomacromolecules due to their fluorescent signals.5
Figure 1
displays the common structure of a WSCP. In this review, only
PTP and conjugate derivatives will be explored in detail.
2. Experimental
2.1 Synthesis of Water Soluble Conjugated Polymers
There are multiple ways of synthesizing WSCP’s. Some
common reactions include palladium-catalysed coup ling
reactions (Suzuki, Heck, and Sonogashira), Wessling reaction,
topopolymerization reaction and FeCl3 oxidative
polymerization.† Depending on which polymer is being
synthesized, certain mechanisms may be better than others. In
this review, the PTP and associated derivative PTPF will be the
† Mechanism outlines see: DOI: 10.1021/cr200263w| Chem. Rev. 2012,
112, 4687−4735

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Fig.1 Common structure of water soluble conjugated polymers6
polymers of interest for describing the mechanism of fluorescent
imaging and cancer treatment.
2.1a PTP Characteristics and Synthesis
The PTP polymer structure is shown in figure 2. It contains
a polythiophene cationic backbone with four distinct
characteristics that make it designed for biomedical applications.
The first one is the low fractional content of porphyrin group s
that are attached to the polythiophene backbone. With a low
content of approximately %1, there comes the benefit of very low
toxicity when photo-excitation doesn’t occur. Second, the
amphiphilic groups are contributing to the promotion of
adsorption to tumor cells by combining electrostatic and
hydrophobic forces. Once the polymers are introduced into the
cancer tissue, it is important they have a strong attachment to
each other so the polymers can be properly monitored. Third, the
porphyrin groups are covalently attached to the polythiophene
backbone which aids in optimizing the FRET process. It also
increases the photocoversion efficiency of singlet oxygen (1
O2)
production, which in turn reduces the light intensit y
requirements of the polymer. These processes will be discussed
later on in the review. The last characteristic is the backbone’s
ability to retain partial emission. This makes the polymer easy
to track and monitor as it triggers apoptosis in the tumor cells.[7,8]
The mechanism of producing PTP is a FeCl3 oxidative
polymerization reaction. The full details of the mechanism can
be seen in the supporting information.†
2.2 Frequency Resonance Energy Transfers (FRET)
Frequency resonance energy transfers are energy transfers
centred on dipole-dipole interactions. They occur between donor
and acceptor molecules that are spatially separated only by a few
nanometres. The molecules that are capable of conducting these
transfers are fluorophores, which can re-emit light once excited
by a light source. In the presence of the acceptor, the donor
molecule will experience a shorter lifetime. In intramolecu lar
FRET, donors and acceptors are connected by a rigid or flexible
linker.9
2.3 Reactive Oxygen Species (ROS)
Reactive oxygen species (ROS) are one of the key factors in
promoting programmed cell death in tumors.
† PTP mechanism see: DOI: 10.1002/adfm.201100840 Adv. Fun. Mat.,
21 (21), 4060
Fig.2 Chemical structure of PTP10
Although some cancer cells may produce ROS themselves,
increasing the activity of the cell to produce excess ROS is the
very aspect that kills them. In earlier studies, it is has been
debated that cancer cells produce more ROS than normal body
cells. This is a hard claim to defend, because it is difficult to find
a comparable “normal” cell to use as a control. The control cell
must replicate some, but not all of the genetic defects in the
tumor cell line. Recent studies have shown that certain
chemotherap eutic agents have the ability of increasing the
oxidant stress in the cell. It is suggested that tumor cells may be
more vulnerable to oxidant stress because they operate with a
heightened level of ROS-mediated signalling, which is required
for growth amongst healthy cells. Although the exact mechanis m
is not known, increasing the oxidant stress in the tumor cells
pushes them beyond their limit of DNA damage and protein
oxidation.11
3. Results and Discussions
3.1 Optical Imaging and Testing of PTP
The objective of the following study was to evaluate the
imaging and therapeutic capability of the PTP polymer. There
were two types of tumor cells that were targeted for cell death:
pulmonary adenocarcinoma cells (A549) and renal cell
carcinoma (A498). Fluorescence microscopy was used to
monitor the structural integrity of the cancer cells exposed to
PTP after certain periods of illumination. Fluorescen ce
microscopy was chosen because it has one of the highest spatial
resolutions compared to other illumination methods. It also beats
nuclear imaging methods because it utilizes nonionizin g
radiation. This is beneficial because it causes the least harm to
the test subject. PTP was exposed to white light between 400-
800 nm and the results were as follows. At 470 nm, the polymer
was excited, but the porphyrin units did not absorb. This non-
absorption leads to emission peaks at 578 nm and 678 nm. It is
important to note that the 678nm peak describes the efficient
energy transfer from the polythiophene backbone to the
porphyrin units. In PTP, the energy transfer significantly
increases the production efficiency of 1
O2 which promotes
apoptosis of the tumor cells.12

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Fig.3 A) 40x magnification of A498 tumor cells under phase contrast bright field and fluorescent field for PTP and EB before and
after 30 minute irradiation. B) Cell viability of cancer cells vs. different durations of light exposure on A498 cells13
For the A498 tumor cells, they were irradiated for 0, 10, and
30 minutes. Ethidium bromide (EB) was used in the study to
make the dead tumor cells easier to identify. Following staining
of the tissue sample, the polymer was irradiated with 470 nm
light which resulted in excitation of the polythiophene backbone.
After the A498 cells were irradiated for an extended time period,
typical apoptotic features began to arise. Some of these changes
include chromatin compaction, condensation of the cytoplasm,
and a large amount of blebbing. Blebbing is described as an
abnormal bulging along the membrane of certain borders of the
cell. When the tumor cells were irradiated for a full 30 minutes,
whole-cell shrinkage was thoroughly observed. Figure 4 display s
the resulting optical images of tumor cells after being irradiated
for the 30 minute time period. In figure 4a, it shows the visible
EB-stained cells from the fluorescent lighting, which indicates
that apoptosis is occurring within the tumor cells as time
progresses. This is consistent with the PTP marked cells that are
visibly excited at 30 minutes. In dark conditions, PTP is emitting
light from the cytoplasm of the tumor cells which indicate the
cells are still alive. However, once irradiation happens, the PTP
is visible only within the nucleus of the tumor cells. Both these
images confirm that as irradiation persists, the tumor cells shrink
to a point where cell death is irreversible.
Figure 4b represents the cytotoxicity of PTP toward A549
cells and A498 cells once irradiated. It is shown that as
irradiation time increases, the viability of the tumor cells
decreases. In other words, the longer the cells are irradiated the
less chance the tumor cells will have recovering their original
functionality. † The cytotoxicity levels of PTP are so low that
surrounding healthy tissue is unaffected by the presence of it in
dark conditions. This is advantageous because there will be no
risk to the subject between the time of applying the PTP and
exposing it to fluorescent light.13
3.2 Optical Imaging and Specificity of PTPF
One of the major issues that researchers encountered with
WSCP’s is the targeting specificity of tumor cells. The tumor
cells that the polymers encounter are negatively charged. The
conjugate polyelectrolytes that incorporate charged groups into
the polymer backbone will bind to the to the cell surface through
electrostatic interactions. The polyelectrolytes certainly help
with increasing the solubility of the conjugated polymer, but the
downside is the selective action of the polymer on a specific cell
is reduced. The way researchers overcame this deterrent was
incorporating groups that removed these electrostatic
interactions and therefore boosting selectivity of the process.
This led to the development of the PTP derivative, PTPF. PTPF
has a slight advantage over its PTP counterpart because it has the
ability to remove these electrostatic forces, resulting in greater
selectivity towards tumor cells. Shown in figure 4a, PTPF is a
charge neutral molecule that is just as soluble as PTP.† It also
has all the optical properties intact that allow it to produce 1
O2
and trigger apoptosis in tumor cells. What’s different about the
PTPF structure compared to the PTP structure is PTPF has folic
acid functionalities incorporated into its polymer backbone. The
folate-receptor (FR) is a receptor that is associated with most
tumor cells. The purpose of using this receptor is that it has a
high affinity for binding to folic acid.14
With this framework in
mind, PTPF will be expected to bind and eliminate tumor cells
with an abundance of folate receptors; since they have had folic
acid fused into them.
Scientists then did a follow-up study with PTPF to test the
specificity and cytotoxic levels towards neighbouring cells. They
used the following tumor cells to test the specificity: KB cells
with high abundance of FRs and NIH-3T3 fibroblast cells that
are FR-negative. To examine how PTPF interacted with the
tumor cells, the researchers took the PTPF and incubated it with
each tumor cell for 24 hours. Figure 4b and 4c represent the
images recorded after the 24 hour period. It is clear that the KB
cells had a greater uptake of the PTPF than the NIH-3T3 cells
mainly due to the FR-folic acid interaction. A greater uptake of
† The standard assay used to test cell viability was the conversion of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl2H-tetrazolium hydro
bromide) into formazan, related to mitochondrial activity. DOI:
10.1002/adfm.201100840 Adv. Fun. Mat., 21 (21), 4061-4062
† PTPF mechanism see: DOI: 10.1002/adfm.201100840 Adv. Fun. Mat.,
21 (21), 4063
A B

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Fig.4 A) Chemical structureof PTPF. B) Fluorescent image of KB cancer cells in the presence of PTPF. C) Fluorescent images of NIH-3T3
cancer cells in thepresence of PTPF. D) Concentration of PTPF vs. cell viability % curves for KB cells (left) and NIH-3T3 cells (right)
treated with PTPF
PTPF is interpreted by the yellow light emitted from the tumor
cells themselves. The specificity of PTPF towards the KB and
NIH-3T3 cells were tested using an MTT assay under 470 nm
light.15
From graph D in figure 4, it is shown that for KB cells,
the longer it is irradiated then the smaller the cell viability will
be. On the contrary, if the KB cells are left in dark conditions,
then PTPF has little to no effect on cell viability. This means that
in the presence of light and PTPF, KB cells will undergo
apoptosis because of the FR-folic acid mediated uptake. In the
right graph in figure 4, it is clear that PTPF had no effect on the
NIH-3T3 tumor under light or dark conditions. This means it had
no cytotoxic effect towards the cancer cells and did not promote
cell death. This is explained by the fact that the NIH-3T3 cells
did not contain the FR receptors as KB cells did. Not having
these receptors means PTPF was not able to interact with the
molecules and therefore not able to trigger 1
O2 generation within
the cell.16
These results lead to the conclusion that when PTPF is
excited by light, the cytotoxicity is specifically more effective
towards tumor cells with high FR abundance than FR-negativ e
tumor cells.
3.3 Nanoparticles in WSCP Structure
Over the past few years, polymers like PTP and PTPF have
been synthesized to continue to help researchers combat certain
diseases like cancer. The structural frameworks of recent
WSCP’s are being innovated in new ways to make up for rising
demands in the biomedical imaging field. The addition of
nanoparticles in WSCP’s is growing in popularity. These
conjugated polymer nanoparticles (CPN) are found to be even
better for biomedical imaging and therapeutic use. This is
because of their high fluorescent brightness, excellent
photostability, and lower cytotoxicity in live-cell imaging.17
Referring to just the imaging aspect, the increased brightness
of light being emitted from the CPN’s allows researchers to use
them at much lower concentrations in cell environments. Using
flow cytometry, the fluorescence emitted from each cell was able
to retain a linear relationship with concentrations as low as 155
pM.18
The other big advantage with CPN’s is the ability to emit
a range of different colours. Cationic 50-100 nm CPN’s have
been developed to display multi-coloured emissions by altering
the FRET energies to a single excitable wavelength. This process
results in different cells in the sample emitting different colours
which makes for efficient marking and identification of tumor
cells.19
One other area where CPN’s are improving is gene and drug
delivery. Nanostructures can be modelled with certain drug or
gene complexes and act as a carrier for the target tissue. These
complexes can be anywhere from 10-100 nm, which makes them
experience less resistance travelling to their destination. The
very low cytotoxicity of these complexes is very beneficial
because they will not harm neighbouring tissue. The cytotoxic
level is so low, it’s like comparing the polymer to the cytotoxic
level of the free drug. The ability of the CPN’s to successfully
deliver a drug or gene to its destination is handy, not to mention
it can also be optimally tracked to make sure it reaches its
target.20
The last application where CPN’s are greatly useful is disease
therapy. It has the same capability as WSCP to kill cancer cells
within a tissue, but it can also eliminate certain bacteria. With the
cationic structures that CPN’s possess, researchers can allow
them to bind to bacteria surfaces and act as a singlet oxy gen
photosensitizer. There are examples where CPN’s have targeted
and eliminated a wide range of Gram-positive and Gram-
negative bacteria.21
Therefore, with all these advantages on the
table and still possessing great potential, CPN’s will be seeing a
lot of research and development over the next decade.
A B
C
D
C

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4. Conclusions
Due to the very high spatial resolution of optical microscop y ,
it has become adherent to the field of water soluble conjugated
polymers. There are many factors that can validate the fact that
WSCPs are ideal for biomedical imaging and therapy. They can
be optically monitored within a system, eliminate tumor specific
cells, and not damage any healthy tissue it comes in contact with.
In PTP, the porphyrin units and polythiophene backbone are able
to undergo FRET’s and efficiently generate 1
O2 to further
promote apoptosis in tumor cells. Whatever PTP lacks in
selectivity, PTPF is able to compensate for it. Utilizing certain
receptors on the tumor cells can motivate researchers to model
polymers so electrostatic forces between polyelectrolytes and
tumor cells are obsolete. With electrostatic forces out of the
equation, WSCP’s will have a much easier chance of pinpointing
a certain tumor grade and destroying it. With the recent discovery
of nanoparticles in conjugated polymers, there is still a lot of
ground to cover in terms of optimizing model framework and
properties. Considering the nanoparticle polymers that scientists
have synthesized thus far are almost identical, if not better, than
previous WSCP’s, it is safe to say the future looks bright for the
field of conjugate polymers.
Acknowledgements
.
The author is grateful towards Professor Greg Welch for the
support and opportunity to write a senior level tutorial review.
Notes and references
a
Dalhousie University, Halifax, NS, Canada
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