Conjugated polymers as multifunctional biomedical platforms: Anticancer activity and apoptosis imaging

Abstract

Recent advances in biological applications of conjugated polymers have focused on highly sensitive diagnostics. The therapeutics of conjugated polymers, however, remains a challenging task. Here we explore for the first time that cationic polythiophene (PMNT) is used as a multifunctional agent for simultaneous cancer therapeutic and apoptosis imaging applications. The anticancer mechanism study showed that the PMNT can uptake inside renal cell carcinoma (A498) cancer cells in a diffusion manner and induce their apoptosis. The increased activation of caspase-3 have been shown to be time- and dose-dependent on PMNT, which indicates a signaling transduction pathway of PMNT induced-apoptosis in A498 cells. Beyond conventional endpoint analysis of apoptosis using multiplex dyes, the PMNT can image the cells and clearly distinguish the living and apoptotic cancer cells. Strikingly, the PMNT could quickly induce cell apoptosis within several minutes under irradiation. The PMNT integrates photosensitivity, anticancer activity and apoptosis imaging, which opens the door for new functional studies of conjugated polymers in disease therapeutics.

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Introduction

Conjugated polymers (CPs) have delocalized electronic structures which exhibit unique electronic and optical properties. These materials have established themselves as useful components in optoelectronic devices as diverse as light-emitting diodes, field effect transistors, photovoltaic cells and organic semiconductor lasers.^1–4 In recent years, water-soluble conjugated polymers have attracted much attention because of their highly sensitive detections for a wide range of biological molecules and processes.^5–9 By taking advantage of the collective amplification behaviors of CPs, trace detection of biomacromolecules in vitro have been extensively studied by us and others.^10–18 Fluorescence imaging in vivo¹⁹ and in cell level^20,21 have also been successfully accomplished using these water-soluble CPs. Very recently, Whitten et al. have initiated a new application of water-soluble CPs as antibiotic agents upon exposure to light.²² The current state of the art in the field focuses on highly sensitive diagnostics using CPs, however few studies have been demonstrated to utilize them in therapeutics.

Recently, extensive research has focused on multifunctional anticancer medicines for simultaneous cancer imaging, diagnosis and therapy, providing a new strategy in cancer treatment.^23,24 Herein we explore for the first time that water-soluble CPs can be used as anticancer agents for simultaneous therapeutic and imaging applications. Our studies demonstrated that the CPs have selective toxicity to different cancer cell lines and can also monitor the apoptosis process of cancer cells by fluorescent imaging. The CP-induced apoptosis of cancer cells was closely related to caspase-3 signaling pathway and have been shown to be time- and concentration-dependent. The present work adds a new dimension to the function of conjugated polymers.

Experimental

Materials and instruments

PFP-G₀,³⁸ PFP-G₀′³⁹ and PMNT³³ were prepared according to the procedures in the literatures. Human embryo lung diploid cell (HPF), pulmonary adenocarcinoma cell (A549), renal cell carcinoma (A498), hepatoblastoma G2 cell (HepG2) and fibroblast cell were purchased from cell culture center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% neonatal bovine serum (NBS), 4.0 mM glutamine and 4500 mg L⁻¹ glucose. NBS was purchased from Sijiqing Biological Engineering Materials (Hangzhou, China). DMEM was purchased from HyClone/Thermofisher (Beijing, China). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) dissolved in 1 × PBS and cytochalasin B in DMSO before use were obtained from Xinjingke Biotech. (Beijing, China). The absorbance for MTT analysis was recorded on a microplate reader (BIO-TEK Synergy HT) at a wavelength of 490 nm. Phase contrast bright-field and fluorescence images were taken with fluorescence microscopy (Olympus 1×71) with a mercury lamp (100 W) as light source.

Cell culture

Five kinds of cells were routinely grown in DMEM (high glucose) medium containing 10% NBS. All cell lines were harvested for subculture using trypsin (0.05%, Gibco/Invitrogen) and grown in a humidified atmosphere containing 5% CO₂ and 95% air at 37 °C.

Cell viability assay by MTT

Cells were subcultured in 96-well plates the day before the experiment at a density of 4∼7 × 10⁴ cells/well, and then cultured for 24 h. The PFP-G₀, PMNT with varying concentrations were respectively added into the cells followed by further culture for 48 h. The culture media were discarded and MTT (1 mg mL⁻¹, 100 μL/well) was added to the wells followed by incubation at 37 °C for 4 h. The supernatant was abandoned, and 150 μL DMSO per well was added to dissolve the produced formazan and the plates were shaken for an additional 10 min. The absorbance values of the wells were then read with microplate reader (BIO-TEK Synergy HT, USA) at a wavelength of 490 nm. The cell viability rate (VR) was calculated according to the following equation:
where the control group was not treated and the experimental group was treated by conjugated polymers. IC₅₀ were analysed by the statistic software SPSS (Version 13.0).

Cells apoptosis experiment with PMNT

The cells were seeded in 35 mm culture plates (Nunc) at a density of approximately 8 × 10⁴ cells/plate. After 24 h, the cells were washed once with 1 × PBS and then grown in 1 mL DMEM medium with and without 150 μM PMNT. Phase contrast bright-field and fluorescence images were snapped at intervals of 12 h using fluorescence microscopy (Olympus 1×71) with 1000 ms exposure time. The false color of PMNT is yellow and the type of light filter is D455/70 nm exciter, 500 nm beamsplitter, and D525/30 nm emitter. The cells were also stained using ethidium bromide (EB) to prove the experiment results using PMNT. The false color of EB is red and the type of light filter is D540/40 nm exciter, 570 nm beamsplitter, and D600/50 nm emitter.

Caspase-3 activity assay

Caspase-3 activities were determined by a quantitative caspase-3 colorimetric assay kit (Keygen, Nanjing, China). The assay is based on spectrophotometric detection of the chromophore after cleavage from the labeled substrate.²⁷ Briefly, A498 cells treated with PMNT and DOX were collected and homogenized in 50 μl cell lysis buffer (containing 0.5 μl DTT), freezing and throwing three times, then centrifuged at 12 000 rpm for 10 min at 4 °C. The supernatant was recovered and the protein concentration was determined by the Bradford method. The 50 μl of the cell lysate corresponding to 200 μg total protein, 50 μl of 2× reaction buffer (containing 0.5 μl DTT) and 5 μl caspase-3 substrate were added to each well of 96-well plates, then the plate was incubated at 37 °C for 4 h. The absorbance value of each well was measured with a microculture plate reader (BIO-TEK Synergy HT, USA) at 400 nm.

Apoptosis of A498 cells by PMNT under light irradiation

The A498 cells were seeded in 35 mm culture plates (Nunc) at a density of approximately 8 × 10⁴ cells/plate for 24 h, and then the cells were washed once with 1 × PBS and then grown in 1 mL DMEM medium with and without 15 μM PMNT. After the cells were further cultured for 12 h, the plates were irradiated under the mercury lamp (100 W) from the microscope for continuous 6 min, where 455/70 nm light attenuated by 75% was used for the irradiation. Phase contrast bright-field and fluorescence images were snapped at intervals of 3 min with fluorescence microscopy (Olympus 1×71). The false color of PMNT is yellow and the type of light filter is D455/70 nm exciter, 500 nm beamsplitter, and D525/30 nm emitter.

Results and discussion

Because most approaches to transfection agents rely on the use of positively charged materials to ensure cellular uptake,²⁵ two cationic conjugated polymers, PFP-G₀ and PMNT were chosen as multifunctional anticancer agents (See Fig. 1a for their chemical structures). Two normal cells, human embryo lung diploid cell (HPF) and fibroblast cell, and three cancer cells, pulmonary adenocarcinoma cell (A549), renal cell carcinoma (A498) and hepatoblastoma G2 cell (HepG2) were used as the target cells in our studies. The cytotoxicities of PFP-G₀ and PMNT to different cell lines were studied by typical MTT assay methods, in which the conversion of soluble MTT into formazan is directly related to mitochondrial activity and subsequently to cell viability.²⁶ As shown in Fig. 1b–c and Table 1, PFP-G₀ with aromatic backbone and ammonium side chain is highly cytotoxic to all the five cells with IC₅₀ values in the range from 22 to 82 μM; PMNT containing sulfur heteroatom represents much more biocompatible except for A498 cell line and can accelerate the growths of fibroblast and A549 cell lines. The selective inhibitions provide basic information that PMNT has an anticancer activity to A498 cells with a IC₅₀ value of 188 μM, thus it shows a potential for renal cancer therapy application.

Fig. 1 (a) Chemical structures of cationic conjugated polymers PFP-G₀ and PMNT. (b–c) Cell viability as a function of polymer concentrations by typical MTT assay. Cells were subcultured in 96-well plates for 24 h the day before the experiment at a density of 4–7 × 10³ cells/well. Then the cells were treated with polymers PFP-G₀ and PMNT with varying concentrations for 48 h, respectively. Error bars represent the standard deviation of three measurements. [PFP-G₀] = 0 ∼ 160 μM, [PMNT] = 0 ∼ 300 μM, [MTT] = 1.0 mg mL⁻¹ (100 μL/well). (d) The caspase-3 relative activity of apoptosis-induced A498 by PMNT and doxorubicin (DOX) as positive control. Error bars represent the standard deviation of three measurements.

Table 1 IC₅₀ (μM) of conjugated polymers to different cells

	fibroblast	HPF	A498	A549	HepG2
a promoting the growth of cell.
PFP-G0	22	21	55	42	82
PMNT	—^a	521	188	—^a	561

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The anticancer mechanism of PMNT was studied by apoptosis experiments. Because the active caspase-3 is a marker for cells undergoing apoptosis,²⁷ we further examined whether the apoptosis of A498 cells in the presence of PMNT directly activated caspase-3. In this experiment, the positive control experiment was also performed using a typical apoptosis agent, doxorubicin (DOX).²⁸ When the A498 cells were treated continuously with PMNT, caspase-3 activity increased 2.2-fold in comparison with that of blank control (Fig. 1d). The increased activation of caspase-3 has been shown to be time- and concentration-dependent on PMNT. The positive control experiment shows that the DOX can also enhance caspase-3 activity in A498 cells under the same experimental conditions as that of PMNT. These results gives clues to a signaling transduction pathway of PMNT-induced apoptosis of A498 cells. It is noted that, although the IC₅₀ value of DOX to A498 cells (0.24 μM) is much lower than that of PMNT, DOX does not show selective apoptosis activity for cancer cells.²⁹

From the results of IC₅₀ or cell viability, we know that the PMNT promotes the growth of fibroblast cells within the testable concentration and is cytotoxic to A498 cells. To get more insights into the interaction mechanism of PMNT with cells, phase contrast and fluorescence images of fibroblast and A498 cells were measured as a function of adding time of PMNT to these cells (Fig. 2 and 3). After culturing for 48 h the density of fibroblast cells with PMNT (Fig. 2b) was obviously larger than that without PMNT (Fig. 2a). The fluorescence images of PMNT likewise represented the living cells, in which karyons were vacant and surrounded by the fluorescence of PMNT within the cytoplasm (Fig. 2c). On the contrary, after 48 h the density of A498 cells with PMNT (Fig. 3b) was obviously smaller than that without PMNT (Fig. 3a). After the addition of PMNT, the A498 cells were continually apoptotic and the density of cells kept close to the initial seeding number, while the control experiment without PMNT showed that the A498 cells were normally going on cell division and proliferation. These phenomena reveal an anticancer mechanism of PMNT that is not cytotoxic to directly damage the cancer cells, but induces their apoptosis at a certain time of cell division cycle. The fibroblast cells are not apoptotic where PMNT should accelerate cell division cycle. As shown in Fig. 3c, the PMNT can image the cells and clearly distinguish the living and apoptotic cancer cells. The vacant fluorescence in the place of nucleus without morphology changes indicates the cell is living. Filling up with dense fluorescence of whole cells and reduced cell size indicates that the cells are dead or in the late stage of apoptosis,^30,31 that is proven by the overlapping locations between the fluorescence of PMNT and the fluorescence of conventional dye, ethidium bromide that only stains dead cells (Fig. 3d).

Fig. 2 Phase contrast bright-field images of fibroblast cells and their fluorescence images in the presence of PMNT in different culturing times. (a) Phase contrast images of fibroblast cells grown without PMNT. (b) Phase contrast images of fibroblast cells grown with PMNT. (c) Fluorescence images of fibroblast cells grown with PMNT at 48 h. Cells were seeded in 35 mm culture plates at a density of approximately 8 × 10⁴ cells per plate. After 24 h, the cells were washed once with 1 × PBS buffer and then grown in 1 mL DMEM medium with and without 150 μM PMNT. Images were snapped at certain time using fluorescence microscopy (Olympus 1×71) with 500 ms exposure time. The false color of PMNT is yellow and the type of light filter is D455/70 nm exciter, 500 nm beamsplitter, and D525/30 nm emitter. The magnification of object lens is 10×.

Fig. 3 (a) Phase contrast images of A498 cells grown without PMNT. (b) Phase contrast images and (c) fluorescence images of A498 cells grown with 150 μM PMNT for 48 h. Images were taken using fluorescence microscopy (Olympus 1×71) with 500 ms exposure time. (d) Fluorescence images of A498 cells using PMNT (left) and EB-stained fluorescence images (right) of A498 cells grown with 150 μM PMNT for 48 h. The fluorescence images were taken with 2000 ms exposure time. The false color of PMNT is yellow and the type of light filter is D455/70 nm exciter, 500 nm beamsplitter, and D525/30 nm emitter. The false color of EB is red and the type of light filter is D540/40 nm exciter, 570 nm beamsplitter, and D600/50 nm emitter. The magnification of object lens is 10×.

Because of the high charge density, it is not possible for the PMNT to freely diffuse across the cell membrane. The uptake mechanism of PMNT into cells was investigated by fluorescence microscopy. To probe whether PMNT was taken up by a passive or active transport mechanism, the A498 cells were incubated with PMNT under low temperature (4 °C). As shown in Fig. 4a, the fluorescence is observed at this temperature, indicating that the PMNT enters cells by a temperature-independent pathway. Since the low temperature can decrease cell membrane fluidity and inhibit the endocytosis process, above results show a non-endocytic mechanism of uptake for PMNT. To confirm this mechanism, cells were co-incubated with PMNT and general endocytosis inhibitor, cytochalasin B that can block the formation of contractile microfilaments and inhibits the endocytosis process.^31,32 As shown in Fig. 4b, the cytochalasin B does not show any inhibition of PMNT uptake.

Fig. 4 (a) Phase contrast bright-field images of A498 cells (left) and their fluorescence images (right) in the presence of 150 μM PMNT under low temperature (4 °C). The images were snapped using fluorescence microscopy (Olympus 1×71) with 1000 ms exposure time. (b) Phase contrast bright-field images of A498 cells (left) and their fluorescence images (right) in the presence of PMNT and cytochalasin B. The cells were pre-cultured in DMEM medium with cytochalasin B (1 μg mL⁻¹) at 37 °C for 2 h and then 150 μM PMNT was added. After 12 h, the cells were washed once with 1 × PBS buffer and images were taken with 300 ms exposure time. The false color of PMNT is yellow and the type of light filter is D455/70 nm exciter, 500 nm beamsplitter, and D525/30 nm emitter. The magnification of object lens is 10×.

We also studied the effect of the ionic polymer's molecular weight on the uptake into cells. Leclerc group's and our attempts to determine the molecular weight of PMNT were not all successful.^33,34 As reported for water soluble polythiophene with a free amino acid side chain or anionic poly(3-alkoxy-4-methyl-thiophene)s prepared by same method as that of PMNT, size exclusion chromatography (SEC) or matrix-assisted laser-desorption ionization time-of-flight spectroscopy (MALDI-TOF-MS) showed that they have number-average molecular weights (M_n) in 6000–10 000 range.^35,36 Here, the PFP-G₀ (M_n = 9000, M_w = 17 100) with lower molecular weight and PFP-G₀′ with higher molecular weight (M_n = 32 970, M_w = 63 400) were used for this purpose. In this experiment, A498 cells was cultured with 10μM PFP-G₀ or PFP-G₀′ for 12 h followed by washing with 1 × PBS buffer. The images were snapped using fluorescence microscopy with 100 ms exposure time. As shown in Fig. 5, the intense fluorescence of PFP-G₀ or PFP-G₀′ in A498 cells shows that the molecular weight of cationic conjugated polymers does not affect their uptake into cells under our experiment conditions.

Fig. 5 The effect of ionic conjugated polymer's molecular weight on the diffusion into A498 cells. Phase contrast bright-field images of A498 cells (left) and their fluorescence images (right) upon treatment with PFP-G₀ or PFP-G₀′. A498 cells were cultured with 10 μM PFP-G₀ (M_n = 9000, M_w = 17 100) or PFP-G₀′ (M_n = 32 970, M_w = 63 400) for 12 h, and then the cells were washed once with 1 × PBS buffer and images were snapped using fluorescence microscopy (Olympus 1×71) with 100 ms exposure time. The type of light filter is D380/30 nm exciter, 420 nm beamsplitter, and D460/50 nm emitter. The magnification of object lens is 10×.

In comparison to the polyfluorene that was studied in this work, polythiophene PMNT always takes on better cell viability (high IC₅₀). The IC₅₀ value is not good enough for an ideal drug. To decrease the IC₅₀ value, we did more experiments and noted that 15 μM of PMNT can kill cancer cells efficiently and quickly (within 6 min) upon exposure to light as comparison to that (150 μM) without exposure to light. The excitation wavelength of irradiation is 455/70 nm and the light source is mercury lamp (100 W) from the microscope (Olympus 1×71) attenuated by 75%. In these experiments, the cells were seeded in 35 mm culture plates at a density of approximately 8 × 10⁴ cells/plate for 24 h, and then the cells were washed once with 1 × PBS buffer and then were grown in 1 mL DMEM medium with or without 15 μM PMNT. After 12 h incubation to ensure the PMNT enters into the cells, the irradiation of A498 with or without PMNT was performed. As shown in Fig. 6a, in the absence of PMNT, the morphology changes of A498 cells are not observed upon the irradiation from 0 to 6 min. While in the presence of PMNT, the cell morphology changes a lot where chromatin compaction, cytoplasm condensation, especially large amount of blebbing and apoptotic bodies are observed (Fig. 6b). It is well-known that the persistent cell volume reduction is a major hallmark of cell apoptosis.³¹ The moving of PMNT fluorescence location from cell cytoplasm to nucleus also indicates the apoptosis of living cancer cells (Fig. 6b insert). It is noted that the apoptosis of A498 cells was not observed upon adding PMNT to the cells and irradiating instantly, which means that the PMNT uptaken inside cells not outside one plays a critical role in inducing apoptosis of A498 cells under the irradiation. The PMNT combines light-harvesting, anticancer activity and apoptosis imaging, which provides new insights on the future design of multifunctional photosensitizer for photodynamic therapy studies.³⁷

Fig. 6 PMNT induces the apoptosis of A498 cancer cells rapidly upon exposure to light. (a) Phase contrast images of A498 cells without PMNT upon the irradiation from 0 to 6 min. (b) Phase contrast images and fluorescence images (insert) of A498 cells treated with 15 μM PMNT for 12 h upon the irradiation from 0 to 6 min. Images were snapped using fluorescence microscopy (Olympus 1×71) with 1000 ms exposure time. The false color of PMNT is yellow and the type of light filter is D455/70 nm exciter, 500 nm beamsplitter, and D525/30 nm emitter. The excitation wavelength of irradiation is 455/70 nm and light source is mercury lamp (100 W) from the microscope attenuated by 75%.

Conclusions

In summary, the selective toxicity of cationic polythiophene derivative to renal cell carcinoma lines A498 and simultaneous apoptosis imaging have allowed us to identify several features that make it an attractive candidate as a multifunctional apoptosis-based therapeutic agent for anticancer study. First, it is the first time that cationic polythiophene (PMNT) is used as multifunctional agent for simultaneous anticancer activity and apoptosis imaging applications. Second, in comparison to conventional endpoint analysis of apoptosis using multiplex dyes, the PMNT has a major advantage in that it can image the cells and clearly distinguish the living and apoptotic cancer cells in view of its brightness and the locations in cells under fluorescence microscopy. Third, the PMNT could accelerate the apoptosis of A498 cancer cells within several minutes upon exposure to light, thus opening the door for new possibilities in the study of conjugated polymer-based photodynamic therapy. Finally, these studies initiate new biological application of conjugated polymers beyond sensing, which opens the door for new functional studies of conjugated polymer in disease therapeutics.

Acknowledgements

The authors are grateful to the National Natural Science Foundation of China (No. 20725308, 90913014, 20721061 and TRR61) and the Major Research Plan of China (No. 2006CB932102).

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