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Tutorial Review

Received 24th March 2014,

Accepted 24th March 2014Light Activated Conjugated Polymers with Anticancer ActivityDavid 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 conjugated 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 (1O2) to further promote apoptosis in tumor cells by method of oxidant stress. The fluorescent properties of these polymers help researchers distinguish between living and dead cells throughout the process by using optical microscopy. 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 development of PTP, scientists were able to create water soluble polymers (such as PTPF) which demonstrate selective anticancer activity towards specific tumor cells.

1. IntroductionWhen 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/energy 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 imaging 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

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There are multiple ways of synthesizing WSCP’s. Some common reactions include palladium-catalysed coupling 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

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 SynthesisThe 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 groups 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 (1O2) production, which in turn reduces the light intensity 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 intramolecular 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 chemotherapeutic 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 mechanism 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 PTPThe 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. Fluorescence 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 nonionizing 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

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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 1O2 which promotes apoptosis of the tumor cells.12

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 displays 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 PTPFOne 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 1O2 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

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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

Fig.4 A) Chemical structure of PTPF. B) Fluorescent image of KB cancer cells in the presence of PTPF. C) Fluorescent images of NIH-3T3 cancer cells in the presence 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 1O2

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-negative tumor cells.

3.3 Nanoparticles in WSCP StructureOver 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

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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 oxygen 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.

4. ConclusionsDue to the very high spatial resolution of optical

microscopy, 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 1O2 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 referencesa Dalhousie University, Halifax, NS, Canada

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