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Sensitive Conjugated-Polymer-Based Fluorescent ATP Probes and Their Application in Cell Imaging Zhifang Chen,†,‡ Pan Wu,†,‡ Rong Cong,§ Naihan Xu,‡ Ying Tan,*,‡ Chunyan Tan,*,†,‡ and Yuyang Jiang‡,∥ †
Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China The Ministry-Province Jointly Constructed Base for State Key Lab, Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China § Cranbrook Kingswood Upper School, Bloomfield Hills, Michigan 48304, United States ∥ Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing 100084, P. R. China ‡
S Supporting Information *
ABSTRACT: Three cationic conjugated polyelectrolytes (CPEs) with a common poly(p-phenylene ethynylene terthiophene) backbone and side chains of different lengths, named as PPET3-N1, PPET3-N2, and PPET3-N3, were designed and synthesized. The UV−vis absorption and fluorescence spectra of the polymers vary strongly with solvent composition, suggesting that the polymers are strongly aggregated in H2O. In addition, the spectroscopic properties of the polymers are affected by small-molecule ATP, characterized by significant fluorescence intensity decreases and red shifts of their absorption bands. Further application of these polymers in cell imaging was studied by confocal fluorescence microscopy, which demonstrated that all of the polymers were localized on the cell membrane and partially inside of cells and that the staining effect gradually increased with the length of the polymer side chains. On the basis of the low cytotoxicity and efficient quenching of PPET3-N2 by ATP, the dose and time effects of ATP on PPET3-N2 imaging were studied, and the results indicated that this polymer might have potential in cell imaging for ATP semiquantification in vivo. KEYWORDS: conjugated polyelectrolytes, ATP, cell imaging, poly(phenylene ethynylene), polythiophene, aggregation
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chemosensors and biosensors5−14 for small molecules, metal ions, proteins, and DNAs on the basis of their amplified fluorescent quenching properties. 15,16 In recent years, CPEs17−29 and polymer dots,30,31 which are composed of conjugated polymers, have become promising alternatives in cell imaging because of their strong fluorescence, high photostability, good cell compatibility, and easy processability. In this work, we designed and synthesized three cationic CPEs that share the same backbone but have side chains of different lengths. Structural and photophysical characterizations of these CPEs were performed, and the interactions of these polymers with a series of negatively charged molecules was studied. Further applications of the three polymers in cell imaging are discussed.
INTRODUCTION As an efficient and versatile investigation approach in the field of biological and medical science, fluorescence cell imaging not only attracts academic attention but also contributes to potential applications. Nowadays, many kinds of organic dyes,1 fluorescent proteins,2,3 and quantum dots (QDs)4 are commercially available for cell imaging. They can adapt to different experimental conditions and possess tunable optical properties. However, some limitations of these current fluorescent materials have become obstacles restricting their applications. For example, some organic dyes suffer from photobleaching and cellular toxicity, fluorescent proteins are often associated with laborious and lengthy procedures, and although QDs have superior brightness and photostability, their cytotoxicity remains a challenge for further applications in vivo. Therefore, making new materials for fluorescent imaging has emerged as a challenging task for chemists and materials scientists. Conjugated polyelectrolytes (CPEs), consisting of electrondelocalized conjugated polymer backbones with ionic side chains, have received considerable attention since the 1990s. They have been widely studied as potential fluorescent © XXXX American Chemical Society
Special Issue: Applied Materials and Interfaces in China Received: July 29, 2015 Accepted: September 15, 2015
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DOI: 10.1021/acsami.5b06935 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces Scheme 1. General Synthetic Route for the Three Polymers and Their Molecular Weight Informationa
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Reagents and conditions: 3:1 (v/v) THF/Et3N, CuI (0.1 equiv), Pd(PPh3)4 (0.038 equiv), reflux.
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24 h. The obtained reaction solution was poured into 200 mL of cold ether, and the red solid was collected and dried (89 mg, 73% yield). 1H NMR (400 MHz, DMSO-d6, δ): 7.34 (dd, 6H), 7.21 (s, 2H), 4.49 (m, 8H), 3.63 (m, 12H), 1.22 (d, 18H). PPET3-N2. PPET3-N2 was synthesized according to the same procedure used for PPET3-N1 except that M2 (105.2 mg, 0.2 mmol) and M4 (100.0 mg, 0.2 mmol) were used. Yield: 83%. 1H NMR (400 MHz, DMSO-d6, δ): 7.37 (ddd, 6H), 7.21 (d, 2H), 4.07 (s, 4H), 3.20 (m, 12H), 3.10 (d, 4H), 1.79 (s, 4H), 1.58 (s, 4H), 1.42 (s, 8H), 1.14 (m, 18H). PPET3-N3. PPET3-N3 was synthesized according to the same procedure used for PPET3-N1 except that M3 (138.8 mg, 0.2 mmol) and M4 (100.0 mg, 0.2 mmol) were used. Yield: 69%. 1H NMR (400 MHz, DMSO-d6, δ): 7.15 (m, 6H), 6.92 (dd, 2H), 3.86 (dt, 8H), 2.98 (12H), 1.55 (s, 4H), 1.32 (s, 8H), 0.85 (m, 46H). Cell Culture. HeLa cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) under a humidified atmosphere of 5% CO2 in air. For all of the experiments, cells were harvested from subconfluent (
