Binding-Directed Energy Transfer of Conjugated Polymer Materials for

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Binding-Directed Energy Transfer of Conjugated Polymer Materials for Dual-Color Imaging of Cell Membrane Qianling Cui, Xiaoyu Wang, Yu Yang, Shengliang Li, Lidong Li, and Shu Wang Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b01424 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 10, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Chemistry of Materials is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials

Binding-Directed Energy Transfer of Conjugated Polymer Materials for Dual-Color Imaging of Cell Membrane Qianling Cui,#a Xiaoyu Wang,#ab Yu Yang,a Shengliang Li,b Lidong Li*a, and Shu Wang*b

#

a

These authors contributed equally to this study.

State Key Laboratory for Advanced Metals and Materials, School of Materials Science and

Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China. b

Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences,

Beijing 100190, P. R. China. Email: [email protected]; [email protected]

1 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Binding of biomolecules or probes to the plasma membrane is of great importance to investigate cell morphology and various biological processes. Herein, a water-soluble conjugated polymer is designed as a membrane probe. The probe shows a strong affinity towards lipid membranes owing to the high charge density from abundant imidazolium moieties together with the moderate rigidity and hydrophobicity derived from the conjugated backbone. Upon binding with a membrane, the inter-chain FRET of the probe was substantially enhanced, which resulted in the emission of both blue and red fluorescence. This is favorable for dual-color imaging. Finally, cellular experiments demonstrate the excellent performance of this macromolecular probe on the stable binding with cell membranes without the appearance of cell endocytocysis even after a long retention time.

Introduction As the first barrier of cells, the plasma membrane serves as the crucial channel to transfer the substances and signals between extra- and intracellular components.1,2 Targeting and binding of biomolecules or probes to the plasma membrane is a common motif in the biological field, owing to its importance in sensing, investigation and manipulation of cellular membranes and their functions.3-6 Generally, to achieve selective accumulation on the plasma membrane, probes are always required to bioconjugate with various recognition elements, which could bind with the complementary receptors overexpressed on the cell membrane through specific antigen–antibody binding or ligand recognition, such as biotin, folate, sialic acid, or rationally designed peptide.7-15 However, despite targeting with the specific ligand–receptor interaction, cellular uptake of these engineered probes always takes place by various endocytosis pathways, especially the nanoparticles.16 In an attempt to achieve binding onto the living cells, the organic probes have been incorporated with a zwitterionic head group and long lipophilic chain, which is structurally similar to phospholipid molecules.17-22 The cell internalization problem has also been encountered for the small organic membrane 2 Environment ACS Paragon Plus

Page 2 of 27

Page 3 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


probe especially after a long retention time due to their poor solubility.17 Thus, challenges remain to develop new probes with efficient targeting ability and stable binding on the cell membrane with comprehensive application. It has been found that the abundant anionic phospholipids in the leaflet of the plasma membrane provides a negatively charged cell surface.23,24 As a result, through electrostatic interactions, the plasma membrane favors binding with polycationic macromolecules, such as peptides with polybasic amino acids, proteins bearing a positively charged surface, or cationic polyelectrolytes. Earlier studies have illustrated that the main driving force for adsorption between oppositely charged lipid membranes and macromolecules is electrostatic neutralization, which is influenced by the amount of charge on the macromolecules.25 Recent experiments have shown that hydrophobicity also plays an important role in complex formation, which aids in insertion of the polymer into the lipid bilayer of the membrane.26,27 The cationic polyelectrolyte with suitable flexibility exhibits a stronger association behavior onto the anionic lipids in a more stretched and flattened conformation, compared with flexible and rigid polyelectrolytes.28 However, numerous cellular experiments have demonstrated that cationic polyelectrolytes with a hydrophobic conjugated backbone migrated across the plasma membrane and accumulated in the cytoplasm, indicating the weak association between the macromolecules and anionic lipids in the membrane.29,30 Moreover, the limited water-solubility of these polymers often caused aggregate formation in an aqueous environment, similar to nanoparticles with a positively charged surface, which exhibited reduced interactions towards the membrane. Thus, good water-solubility is also necessary to avoid cellular uptake, ensuring the polymer exists as a unimolecular chain to fully and firmly contact with the lipids on the plasma membrane. In this work, we design and synthesize a cationic probe based on a water-soluble conjugated polymer with efficient binding onto the plasma membrane. The side chains bear abundant imidazolium groups with a high positive charge density and oligo(ethylene glycol) 3 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 27

(EG) moieties, providing electrostatic binding sites and sufficient hydrophilicity. The conjugated polymer serves as the rigid and hydrophobic part, which along with the fluorophore, undergoes fluorescence resonance energy transfer (FRET) to indicate the polymer binding state. This probe is proven to stably associate with the model lipid vesicles and plasma membrane, while its bright blue/red fluorescence signal also enables it to be a promising membrane probe for dual-color imaging. Our results provide new insights for the designing and development of probes in the controlled recognition of and adsorption on the plasma membrane.

Experimental Section Materials.

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene

(monomer

A),

2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-bis(6′-bromohexyl)fluorene (monomer B), and BIMEG were synthesized according to the procedure found in the literatures.31-33 benzothiadiazole

Tetrakis(triphenylphosphine)palladium(0) (DBT),

diethylene

glycol

(Pd(PPh3)4),

2-bromoethyl

1,4-dithienyl

methyl

ether,

di(1H-imidazol-1-yl)methane, tetrabutylammonium chloride, potassium hexafluorophosphate, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine

perchlorate

(DiD),

and

1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DMPG) were purchased from Sigma–Aldrich and used as received. Other chemicals solvents were purchased from Beijing Chemical Works and used without further purification. Ultrapure Millipore water (18.6 MΩ cm) was used throughout the experiments. Synthesis of PF-DBT-Br. Monomer A (0.585 g, 0.9 mmol), monomer B (0.744 g, 1.0 mmol), and DBT (0.046 g, 0.1 mmol) were dissolved in a mixture of toluene (25 mL) and K2CO3 (2 M, 5 mL) with a molar ratio of 45:50:5. After degassing with argon for 30 min, the catalyst Pd(PPh3)4 was added. The reaction was covered with an aluminum foil and stirred at 85 °C for 48 h under an argon atmosphere. Then, the mixture was extracted by chloroform 4 Environment ACS Paragon Plus

Page 5 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and concentrated. The desired polymer was isolated by triply re-precipitating from chloroform to acetone and dried under vacuum, obtained as a red powder (0.41 g, 30%). 1H NMR (200 MHz, CDCl3, δ): 7.50–7.83 (br, aromatic backbone), 3.26–3.28 (br, CH2Br), 0.83–2.39 (br, NCH2(CH2)6). GPC: Mn=15904, Mw=23696, PDI=1.49. Synthesis of PF-DBT-BIMEG. PF-DBT-Br (0.10 g) and BIMEG (1.20 g, 4 mmol) were dissolved in 20 mL DMF and stirred at 80 °C for 48 h under argon. Then the solvent was removed by vacuum distillation and the residue was dissolved in water. Potassium hexafluorophosphate was added to the solution and the precipitate was filtered. The solid was dissolved in acetonitrile and precipitated by addition of tetrabutylammonium chloride. The resulting product was dissolved in dimethyl sulfoxide (DMSO)/water and dialyzed against water for one week. Then, the red polymer was obtained after freeze drying. 1H NMR (200 MHz, DMSO, δ): 8.10–8.60 (br, imidazolium), 7.70–8.10 (br, aromatic backbone), 6.75–7.05 (br, NCH2N), 4.30–4.45 (br, NCH2(CH2)6), 4.00–4.20 (br, NCH2CH2O), 3.70–3.90 (br, OEG), 0.90–1.70 (br, NCH2(CH2)6). Synthesis of PF-DBT. PF-DBT-Br (0.10 g) was dissolved into 50 mL of chloroform, and added by trimethylamine solution in ethanol (30%, 5 mL). The mixture was stirred for 48 h at room temperature. The solvent was evaporated by a rotary evaporator and the solid was dissolved in DMSO/water and dialyzed against water for one week. Then, the red polymer was obtained after freeze drying. 1H NMR (200 MHz, DMSO, δ): 7.55–8.10 (br, aromatic backbone), 4.00–4.20 (br, CH2Br), 3.10–3.50 (br, N(CH3)3), 0.90–1.60 (br, NCH2(CH2)6). Cytotoxicity Assay by MTT Method. The cytotoxicity of PF-DBT-BIMEG against HeLa

and

MCF-7

cells

were

studied

using

a

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. HeLa or MCF-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). Then, the cells were seeded into 96-well plates at a density of 7 × 103 cells in each well. After 24 h of incubation at 37 °C in a 5% CO2 5 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

humidified

atmosphere,

the

cells

were

treated

with

Page 6 of 27

various

concentrations

of

PF-DBT-BIMEG aqueous solutions (0–100 µg/mL), and cultured for another 24 h. After pouring out the medium, 100 µL of freshly prepared MTT (1 mg/mL in PBS) was added to each well and incubated for 4 h. After removing the MTT medium solution, the cells were lysed by adding 100 µL of DMSO. The plate was gently shaken for 5 min, and then the absorbance of purple formazan at 570 nm was monitored using a Spectra MAX 340PC plate reader. Cytotoxicity Assay by CCK-8 Method. The cytotoxicity of PF-DBT-BIMEG against Jurkat suspension cells were studied using a general protocol of cell counting CCK-8 assay. Jurkat cells were seeded into 96-well plates at a density of 7 × 103 cells in each well. After 24 h of incubation at 37 °C in a 5% CO2 humidified atmosphere, the cells were incubated with various concentrations of PF-DBT-BIMEG aqueous solutions (0–100 µg/mL), and cultured for 24 h. Then, 10 µL of CCK-8 solution was added to each well followed by incubation for another 4 h. Cellular Imaging Experiments. HeLa, MCF-7, and Jurkat cells were seeded in 35 × 35 mm culture plates and maintained for 24 h in DMEM containing 10% (v/v) fetal bovine serum (FBS) in a humidified incubator with 5% CO2 atmosphere at 37 °C. These cells were respectively incubated with PF-DBT-BIMEG at 50 µg/mL in PBS solution for 30 min, and then incubated with DiD at 5 µM in PBS solution for another 15 min. Then, the medium was removed and the cells were washed with PBS, and the fluorescence images and phase contrast bright-field images were recorded on a confocal fluorescence microscope (Olympus FV1000-IX81). For observing the signals of PF-DBT-BIMEG, the excitation wavelength was set at 405 nm and collection of fluorescence signals from 425 to 525 nm (blue channel) and 575–625 nm (red channel). The excitation wavelength used for DiD is 635 nm, and the fluorescence signals were collected in the range of 655–715 nm.


Page 7 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Preparation of Lipid Vesicles. Large unilamellar vesicles (LUVs) were prepared by the extrusion method as previously reported.34,35 Briefly, 3 mg of phospholipid DMPG were dried by evaporation under a dry nitrogen gas stream and then under vacuum for 3h. Lipid vesicles were formed by resuspending the dried DMPG in water to a final concentration of 0.5 mM and under ultrasonication for 10 min. Then, the vesicle suspension was extruded through 0.22 µM poly(tetrafluoroethylene) (PTFE, Millipore) filter. Characterization.

1

H NMR spectra were recorded on a 200 MHz AC Bruker

spectrometer, using CDCl3 or d6-DMSO as the solvents. Elemental analysis was conducted on a FLASH EA 1112 element analyzer. Gel permeation chromatography (GPC) analysis was carried out on a Waters Styragel system using polystyrene as the calibration standard and tetrahydrofuran as eluent. The hydrodynamic diameters and zeta potentials of the particles were determined using a Nano ZS90 Zetasizer (Malvern Instruments Ltd., UK). The UV-VIS absorption spectra were measured on a Hitachi U3900 spectrophotometer. Fluorescence spectra of the samples were recorded by a Hitachi F-7000 spectrometer. The absolute fluorescence quantum yields were determined using a spectrofluorometer (NanologR FluoroLog-3-2-iHR320, Horiba Jobin Yvon) equipped with an integrating sphere. The excitation wavelength was set at 380 nm. The scattering spectral range of the blank and sample was set from 375 nm to 385 nm, and the emission spectral range was from 392 to 750 nm. The maximal absorption intensity of the sample solution was controlled to be less than 0.10, and the same volume of the solvent was used as the blank sample. Time-domain lifetime measurements were performed using an Edinburgh Instruments F900 spectrometer with an excitation wavelength of 380 nm. Scanning electron microscopy (SEM) images were captured on a field-emitting scanning elctron microscope (FESEM, JEOL JSM-7401F).

Results and Discussions



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Design, Synthesis, and Characterization of PF-DBT-BIMEG. Imidazolium and its derivatives with a positive charge have been reported as selective receptors for the negatively charged species especially biomolecules, not only through the strong electrostatic attraction, but also derived from the special hydrogen bonds between the C2-H and anions.33,36,37 It might be a good candidate to interact with the negatively charged plasma membrane. Herein, bis-imidazolium (BIM) groups with high charge density were selected as binding sites with anionic lipids, whereas the EG moieties were also involved to further improve water solubility, and reduce undesirable aggregation and nonspecific interactions with biomacromolecules. However, as the fluorophore and hydrophobic part, a conjugated backbone was selected considering their outstanding optical properties, such as high fluorescence quantum yields, good photostability, and nontoxicity, which provides a great deal of interest for bioimaging applications in vitro and in vivo.38-42 In this work, we choose a conjugated polymer backbone composed of poly(fluorene) (PF) doped with 1,4-dithienylbenzothiadiazole (DBT) units at 5 mol%, serveing as the donor and acceptor for FRET, respectively. The intra-chain FRET efficiency of PF-DBT-BIMEG is low owing to the low doping ratio of DBT, while inter-chain FRET would be enhanced upon aggregation (Scheme 1), which could be employed to indicate the polymer binding with the cell membrane.

Scheme 1. Schematic representation of aggregation-induced inter- and intra-molecular FRET from PF segments to DBT units on the cell membrane.


Page 8 of 27

Page 9 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The synthetic route of the polymer is displayed in Scheme 2. The polymer precursor (PF-DBT-Br) was synthesized by Suzuki cross coupling reaction using fluorene monomer A and B, doped with DBT monomer at 5 mol%. Its average molecular weight (Mw) determined by GPC was 23 696 g mol-1 with a polydispersity index (PDI) of 1.49. BIMEG was prepared in our previous work, where diethylene glycol 2-bromoethyl methyl ether and di(1H-imidazol-1-yl)methane were heated together at 80 °C in acetonitrile for 2 days under argon.31 Then, the polymer precursor reacted with excessive BIMEG in DMF at 80 °C for 2 days under argon atmosphere, giving rise to the target polymer PF-DBT-BIMEG. The polymer was chracterized by NMR spectroscopy (Figure S1) and elemental analysis (Table S1), and found to be in good agreement with the structures. The molar ratio of the DBT units in the PF-DBT-Br was calculated to be 4.7% and 5.0% for measurement 1 and 2, respectively, based on the N/C ratio of the elemental anaylsis result, which is close to the feeding ratio. In addition, the N/C ratio of the PF-DBT-BIMEG was comparable to the theoretical value, proving the nearly complete substituation of BIMEG groups on the side chains. Moreover, the resulted polymer can be directly dissolved in water, proving its good water-solubility.

Scheme 2. Synthesis route of PF-DBT-BIMEG.

Photophysical Properties of PF-DBT-BIMEG. The photophysical properties of PF-DBT-BIMEG were investigated in water. The absorption curve in Figure 1a displayed a



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

maximum absorption at 398 nm and a weak and broad absorption band at 450–600 nm, which was attributed to the PF segments and DBT units, respectively. Compared to the precursor (PF-DBT-Br) having a absorption peak around 384 nm in dichloromethane (Figure S2), the PF absorption of PF-DBT-BIMEG in water showed a 15 nm red-shift due to the solvent effect. Upon excited with 380 nm irradiation, PF-DBT-BIMEG exhibited a intense emission peak at 423 nm from the PF segments with a shoulder peak at 445 nm. Meanwhile, the low-bandgap fluorophore, DBT, showed a relatively low fluorescence emission band at 560–750 nm with an excitation wavelength of 380 nm. It is well known that the spectroscopic overlap between the emission of the donor and the absorption of the acceptor, together with suitable distance between them (less than 10 nm), are the two prerequisites to achieve efficient FRET.43 The spectral overlap between the emission of PF and absorption of DBT implies the possibility of FRET from PF to DBT. To prove this assumption, the excitation spectra of PF-DBT-BIMEG were recorded with an emission wavelength at 420 nm and 655 nm, respectively. For DBT emission (655 nm), there were two excitation peaks around 390 and 530 nm. The former peak at 390 nm is consistent well with the absorption of PF moiety in Figure 1a and the excitation spectrum of PF emission (420 nm), indicating the occurrence of intra-molecular energy transfer from PF to DBT. However, this FRET efficiency is quite low because of the low molar ratio of DBT and the large spatial distance between the energy pair. Figure 1c shows the fluorescence spectra of PF-DBT-BIMEG at different pH values, where almost no obvious change was found in the investigated range (pH=4–8). This indicates that the emission of PF-DBT-BIMEG is stable against pH variation, which is favorable for application in the diverse intracellular pH environment. Moreover, the absolute fluorescence quantum yield of PF-DBT-BIMEG in water was measured to be 24.2%. To study the molecular state in water, the PF-DBT-BIMEG aqueous solution was examined by dynamic light scattering, with a distribution of less than 1 nm observed (Figure 1d), which can be ascribed to the hydrodynamic diameter of an individual polymer chain. As 10 Environment ACS Paragon Plus

Page 10 of 27

Page 11 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a control, PF-DBT with the same backbone bearing quaternary ammonium salts was synthesized (Figure S3, S4) shows a broad distribution range from 100 nm to 1000 nm (Figure S5), indicating the existence of large aggregates. This further proves that the abundant positive charges on BIM groups and hydrophilic EG moieties enhance the water solubility although the backbone is strongly hydrophobic. The unimolecular state in water is also responsible for the low FRET efficiency from PF to DBT since inter-chain FRET is negligible.

Figure 1. (a) Normalized UV-VIS absorption (black curve) and fluorescence emission spectrum (red curve) of PF-DBT-BIMEG in water. The excitation wavelength is 380 nm. (b) Normalized excitation spectra of PF-DBT-BIMEG aqueous solution with emission at 420 nm (black curve) and 655 nm (red curve), respectively. (c) Fluorescence spectra of PF-DBT-BIMEG aqueous solutions with different pH values. (d) The hydrodynamic diameter of PF-DBT-BIMEG chains in water measured by dynamic light scattering.

To investigate the interaction of the polymer under a lipophilic environment in a controlled manner, spectroscopic analyses were carried out in the presence of large 11 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

unilamellar

vesicles

(LUVs)

Page 12 of 27

composed

of

1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DMPG) (Figure 2a). After incorporation of PF-DBT-BIMEG, the zeta potential of these vesicles changed from –25.2 mV t o –6.3 mV, which proved the successful loading of the polyelectrolytes. Meanwhile, the reduction in the emission band of PF and the enhancement in the emission peak of DBT clearly show the efficient FRET between the two segments, which is directly demonstrated by the visual fluorescence color change from purple to red (Figure 2b). It’s should be noted that the distinct vibronic shifts observed in the emission band of the PF backbone implies that mechanical chain stretching occurs in the electrostatic complex.44 Considering that the electrostatic interaction is easily screened by higher ion strength, herein, the salt concentration was increased to determine if the binding in this complex would be reduced. Interestingly, only a slight change in fluorescence intensity and ratiometric signal was observed (Figure S6), indicating that the electrostatic neutralization is not the main force between the two populations. Moreover, we assumed that this firm binding was derived from the immobile polymer nanodomains of PF-DBT-BIMEG assembled on lipid vesicles. The lipid mobility is decreased significantly by the adsorbed polyelectrolyte (attractive interactions with multivalent polymer nanodomains), which hopefully reduces the cell internalization through the endocytic pathway. To further understand the change in optical properties, the fluorescence lifetime of the polymer was examined in the absence and presence of LUVs. Fluorescence decay curves in Figure 2c and 2d show that the emission at 420 nm decays faster than before, while the emission at 655 nm displays an inverse tendency. The reduction in the calculated average lifetime (from 0.55 ns to 0.23 ns, Table S2) at 420 nm peak can explain the decrease in PF emission intensity through energy transfer to DBT units. On the other hand, the fluorescence enhancement was observed for the DBT emission at 655 nm in the presence of LUVs excited by 550 nm wavelength (Figure S7). This phenomenon implies the increase in intrinsic 12 Environment ACS Paragon Plus

Page 13 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


quantum yield of DBT due to a reduced non-radiative rate caused by the change of local environment from hydrophilic water to relatively hydrophobic lipid membrane. It can also explain the longer lifetime of DBT emission in the presence of LUVs. Thus, we can attribute the observed increased DBT emission at 655 nm to both FRET from PF to DBT and increased quantum yield of the DBT moiety. To make a more quantitative estimate, the lifetime changes by about 30% would account for a similar change in emission intensity. Hence the major change of acceptor emission intensity, which is about a factor of 2.5, is due to FRET.

Figure 2. (a) Schematic illustration of lipid vesicles formed by DMPG and interaction with PF-DBT-BIMEG. (b) Emission spectra of PF-DBT-BIMEG aqueous solution in the absence and presence of DMPG lipid vesicles. Insets are the corresponding photographs of PF-DBT-BIMEG and PF-DBT-BIMEG+LUVs solutions under a hand-held UV lamp illumination. Decay curves of 420 nm emission wavelength (c) and 655 nm emission wavelength (d) of PF-DBT-BIMEG in the absence and presence of LUVs.

Cell Membrane Imaging Assay. Subsequently, the cell imaging experiments were carried out for human cervical carcinoma cell (HeLa) incubation with PF-DBT-BIMEG at a



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

concentration of 50 µg/mL for 30 min. A commercial cell membrane dye, DiD, was also co-cultured with these cells to indicate the location of polymer staining. After the culture media was removed and washed with PBS buffer, cells were observed under a confocal laser scanning

microscope

(CLSM).

The

excitation

laser

wavelength

for

observing

PF-DBT-BIMEG and DiD was set at 405 nm and 635 nm, respectively. The obtained CLSM images are shown in Figure 3a. Both of images acquired from blue and red channels displayed a clear outline of the tested living cells, implying the location of PF-DBT-BIMEG in the cell membrane. Furthermore, the well merged imaging pattern with that of DiD (gray color was used instead of red to distinguish between the red signals of the polymer) also demonstrates the specific location of PF-DBT-BIMEG in the cell membrane. Besides, the overlapped 3D CLSM fluorescence image of HeLa cells (Figure 3b) indicates that the blue and red fluorescence signals arise from the surface of cells, further suggesting that the cell membrane is stably labeled by PF-DBT-BIMEG. To investigate if the polymer enters into the cell interior after a longer time through an endocytosis process, the cell imaging was also performed after 24 h incubation with HeLa cells. As shown in Figure 3a, there is almost no obvious difference within the studied period of 24 h, except an enhancement in fluorescence intensity arising from the accumulation of polymers towards the cell membrane, proving the stable staining performance of PF-DBT-BIMEG towards the cell membrane. By contrast, no clear cell membrane imaging or cell imaging was observed for PF-DBT after 30 min incubation (Figure S8). After 24 h incubation, the cell imaging result is not as good as that observed for PF-DBT-BIMEG. Some PF-DBT chains entered into the cell cytoplasm, and the undesirable aggregation and non-specific adsorption with a plastic substrate was also observed. Thus, the BIMEG side chains on PF-DBT-BIMEG are thought be responsible for its specific cell membrane targeting and long retention time. The cytotoxicity is also a crucial parameter to evaluate a probe for application in living cells. After incubation with PF-DBT-BIMEG for 24 h, the MTT result 14 Environment ACS Paragon Plus

Page 15 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shows the cell viabilities over 90% in the tested concentration range of the polymer (Figure 3c).

Figure 3. (a) Confocal laser scanning microscopy (CLSM) images of HeLa cells stained with PF-DBT-BIMEG (50 µg/mL) and DiD dyes for 30 min and 24 h. The excitation laser wavelength for PF-DBT-BIMEG and DiD is 405 nm and 635 nm, respectively. The scale bars represent 20 µm. (b) The overlapped 3D CLSM fluorescence image of HeLa cells after incubation with PF-DBT-BIMEG (50 µg/mL) for 30 min. (c) Cell viability of HeLa cell as a function of PF-DBT-BIMEG concentration.

To study the molecular state of PF-DBT-BIMEG on the cell membrane, the fluorescence spectra were collected from different sites on cells by CLSM under excitation with a 405 nm laser. Here, only the blue channel was used for the cell imaging, giving rise to the blue fluorescence image in Figure 4. After 30 min incubation with HeLa cells, the fluorescence ratiometric measurements (I620/I425) of the probe varied in the range from 0.36 to 0.45, which is higher than that of free states in aqueous solution (0.13) (Figure 4a). The strong electrostatic attraction between highly positive PF-DBT-BIMEG and negative cell membrane



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

causes polymer aggregation, which increases the local concentration of DBT units and subsequently enhances the FRET efficiency from PF to DBT units. After 24 h incubation, an obvious further increase in the fluorescence ratiometric measurements (I620/I425) within the range of 0.90–1.10 was observed (Figure 4b), consistent with the enhanced brightness of the red channel observed in Figure 2b. As the incubation time increased, the accumulation and inter-attraction of polymer aggregates in the cell membrane further shorten the intra-molecular and inter-molecular distances between the donor and acceptor, giving rise to a significant enhancement of the FRET efficiency, and consequently, the red fluorescence intensity. The comparable intensity of blue and red emission make the PF-DBT-BIMEG an ideal staining agent for dual channel cellular imaging. To investigate the morphology of the macromolecular dye on the cell membrane, the HeLa cells co-cultured with PF-DBT-BIMEG were observed using scanning electron microscopy (SEM). The blank HeLa cells in Figure 4c display a clear and rough surface. After incubation with PF-DBT-BIMEG for 24 h, the cell surface was uniformly coated with a polymer layer with a high surface coverage as revealed by SEM images (Figure 4d). This further confirmed the stable binding of PF-DBT-BIMEG towards the plasma membrane.


Page 16 of 27

Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Fluorescence spectra collected from different sites on the cell membrane of HeLa cells after incubation with PF-DBT-BIMEG for 30 min (a) and 24 h (b), obtained by confocal laser scanning microscopy. The insets show the corresponding collecting sites on the cell membrane. SEM image of blank HeLa cells (c) and HeLa cells after incubation with PF-DBT-BIMEG for 24 h (d).

Furthermore, to test if the probe is capable of membrane imaging of other kinds of living cells, breast cancer cells (MCF-7) and suspension cells (Jurkat) were choosed to incubated with PF-DBT-BIMEG for 30 min. The obtained CLSM images in Figure 5a show the clear outlines of the cells and the well overlap with the DiD images, proving the probe can stained these two cell lines effectively and specifically. In addition, the MTT result in Figure 5b indicats nearly 100% cells were alive in the tested concentration range. Thus, PF-DBT-BIMEG is very safe to be used in living cells as an comprehensive fluorescent membrane marker. The common strategy to realize dual-color imaging is to integrate two fluorophores into a nanoparticle or system, which is complex and time-consuming.45-48 Here, the bright blue/red dual emission under excitation at a single wavelength was realized by a simple water-soluble 17 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

polymer through binding-directed energy transfer on membrane. Besides, compared to small molecule membrane probes, the fluorescent conjugated polymers show good stability and water-solubility, together with large Stokes shift due to FRET process. More importantly, almost no any cell endocytosis was observed for this polymeric probe, which was often found for small organic probes as membrane marker.20 Therefore, we believe this polymeric probe is an ideal cell membrane probe for biological application.

Figure 5. (a) Confocal laser scanning microscopy (CLSM) images of MCF-7 cells (up) and Jurkat cells (down) stained with PF-DBT-BIMEG (50 µg/mL) and DiD dyes for 30 min. The excitation laser wavelength for PF-DBT-BIMEG and DiD is 405 nm and 635 nm, respectively. The scale bars represent 20 µm. (b) Cell viability of MCF-7 and Jurkat cell lines as a function of PF-DBT-BIMEG concentration.

Conclusions In summary, a novel water-soluble conjugated polymer carrying abundant imidazolium moieties and an intrinsic intra-FRET backbone was designed and synthesized as a stable 18 Environment ACS Paragon Plus

Page 18 of 27

Page 19 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


membrane probe. The strong electrostatic attraction between imidazolium groups and negatively charged lipids drives the association of the polymer with the cell membrane, and the conjugated backbone provides a suitable hydrophobicity and rigidity. Cellular imaging experiments demonstrated that PF-DBT-BIMEG quickly, specifically and effectively stain cell membranes in many different cell lines. In addition, aggregation-enhanced FRET on the cell membrane enhances the red emission, further proving that it is a promising candidate as a cell-membrane-located probe with dual-color imaging.

ASSOCIATED CONTENT Supporting Information This Supporting Information is available free of charge on ACS Publications website. Additional characterization data of PF-DBT-Br (NMR spectra, elemental analysis, absorption and emission spectra), synthesis and characterization data of PF-DBT (synthetic scheme, absorption/emission spectra, DLS, cell viability and imaging), additional characterization data of PF-DBT-BIMEG (element analysis, salt effect on emission, and fluorescence lifetime).

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected] Author Contributions Q.C. and X.W. contributed equally to this study. Notes The authors declare no competing financial interest.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACKNOWLEDGEMENTS The authors are grateful to the National Natural Science Foundation of China (51373022, 51503015), the General Financial Grant from the China Postdoctoral Science Foundation (2015M581176). We acknowledge Prof. Dr. Helmuth Möhwald (Max Planck Institute of Colloids and Interfaces, Germany) and Prof. Dr. Yuxia Zhao (Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, China) for his/her useful discussion.

REFERENCES (1)

Schlessinger, J.; Axerlrod, D.; Koppel, D. E.; Webb, W. W.; Elson, E. L. Lateral Transport of a Lipid Probe and Labeled Proteins on a Cell Membrane. Science 1977, 195, 307–309.

(2)

Lingwood, D.; Simons, K. Lipid Rafts as a Membrane-Organizing Principle. Science 2010, 327, 46–50.

(3)

Demchenko, A. P.; Mély, Y.; Duportail, G.; Klymchenko, A. S. Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes. Biophysical. J. 2009, 96, 3461–3470.

(4)

Cairo, C. W.; Key, J. A.; Sadek, C. M. Fluorescent Small-Molecule Probes of Biochemistry at the Plasma Membrane. Curr. Opin. Chem. Bio. 2010, 14, 57–63.

(5)

Klymchenko, A. S.; Kreder, R. Fluorescent Probes for Lipid Rafts: From Model Membranes to Living Cells. Chem. Biol. 2014, 21, 97–113.

(6)

Honigmann, A.; Mueller, V.; Ta, H.; Schoenle, A.; Sezgin, E.; Hell, S. W. Scanning STED-FCS Reveals Spatiotemporal Heterogeneity of Lipid Interaction in the Plasma Membrane of Living Cells. Nat. Commun. 2014, 5, 5412–5423.

(7)

Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J.; Yan, H.; Fan, C. Single-Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells. Angew. Chem. Int. Ed. 2014, 53, 7745–7750. 20 Environment ACS Paragon Plus

Page 20 of 27

Page 21 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(8)

Wu, C.; Schneider, T.; Zeigler, M.; Yu, J.; Schiro, P. G.; Burnham, D. R.; McNeill, J. D.; Chiu, D. T. Bioconjugation of Ultrabright Semiconducting Polymer Dots for Specific Cellular Targeting. J. Am. Chem. Soc. 2010, 132, 15410–15417.

(9)

Rong, Y.; Wu, C.; Yu, J.; Zhang, X.; Ye, F.; Zeigler, M.; Gallina, M. E.; Wu, I.; Zhang, Y.; Chan, Y.-H.; Sun, W.; Uvdal, K.; Chiu, D. T. Multicolor Fluorescent Semiconducting Polymer Dots with Narrow Emissions and High Brightness. ACS Nano 2013, 7, 376–384.

(10)

Tang, F.; Wang, C.; Wang, X.; Li, L. Facile Synthesis of Biocompatible Fluorescent Nanoparticles for Cellular Imaging and Targeted Detection of Cancer Cells. ACS Appl. Mater. Interfaces 2015, 7, 25077–25083.

(11)

Li, M.; Nie, C.; Feng, L.; Yuan, H.; Liu, L.; Lv, F.; Wang, S. Conjugated Polymer Nanoparticles for Cell Membrane Imaging. Chem. Asian J. 2014, 9, 3121–3124.

(12)

Wang, B.; Zhu, C.; Liu, L.; Lv, F.; Yang, Q.; Wang, S. Synthesis of a New Conjugated Polymer for Cell Membrane Imaging by Using an Intracellular Targeting Strategy. Polym. Chem. 2013, 4, 5212–5215.

(13)

Matsumoto, A.; Sato, N.; Kataoka, K.; Miyahara, Y. Noninvasive Sialic Acid Detection at Cell Membrane by Using Phenylboronic Acid Modified Self-Assembled Monolayer Gold Electrode. J. Am. Chem. Soc. 2009, 131, 12022–12023.

(14)

Liu, A.; Peng, S.; Soo, J. C.; Kuang, M.; Chen, P.; Duan, H. Quantum Dots with Phenylboronic Acid Tags for Specific Labeling of Sialic Acids on Living Cells. Anal. Chem. 2011, 83, 1124–1130.

(15)

Shinde, S.; El-Schich, Z.; Malakpour, A.; Wan, W.; Dizeyi, N.; Mohammadi, R.; Rurack, K.; Wingren, A. G.; Sellergren, B. Sialic Acid-Imprinted Fluorescent Core−Shell Particles for Selective Labeling of Cell Surface Glycans. J. Am. Chem. Soc. 2015, 137, 13908–13912.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(16)

Zhang, S.; Gao, H.; Bao, G, Physcial Principles of Nanoparticle Cullular Endocytosis. ACS Nano 2015, 9, 8655–8671.

(17)

Heinrich, L.; Freyria, A.-M.; Melin, M.; Tourneur, Y.; Maksoud, R.; Bernengo, J.-C.; Hartmann, D. J. Confocal Laser Scanning Microscopy Using Dialkylcarbocyanine Dyes for Cell Tracing in Hard and Soft Biomaterials. J. Biomed. Mater. Res., Part B. 2007, 81, 153–161.

(18)

Kucherak, O. A.; Oncul, S.; Darwich, Z.; Yushchenko, D. A.; Arntz, Y.; Didier, P.; Mély, Y.; Klymchenko, A. S. Switchable Nile Red-Based Probe for Cholesterol and Lipid Order at the Outer Leaflet of Biomembranes. J. Am. Chem. Soc. 2010, 132, 4907–4916.

(19)

Zhang, X.; Wang, C.; Jin, L.; Han, Z.; Xiao, Y. Photostable Bipolar Fluorescent Probe for Video Tracking Plasma Membranes Related Cellular Processes. ACS Appl. Mater. Interfaces 2014, 6, 12372–12379.

(20)

López-Duarte, I.; Vu, T. T.; Lzquierdo, M. A.; Bull, J. A.; Kuimova, M. K. A Molecular Rotor for Measuring Viscosity in Plasma Membranes of Live Cells. Chem. Commun. 2014, 50, 5282–5284.

(21)

Kreder, R.; Oncul, S.; Kucherak, O. A.; Pyrshev, K. A.; Real, E.; Mély, Y.; Klymchenko, A. S. Blue Fluorogenic Probes for Cell Plasma Membranes Fill the Gap in Multicolour Imaging. RSC Adv. 2015, 5, 22899–22905.

(22)

Collot, M.; Kreder, R.; Tatarets, A. L.; Patsenker, L. D.; Mély, Y.; Klymchenko, A. S. Bright Fluorogenic Squaraines with Tuned Cell Entry for Selective Imaging of Plasma Membrane vs. Endoplasmic Reticulum. Chem. Commun. 2015, 51, 17136–17139.

(23)

McLaughlin, S.; Murray, D. Plasma Membrane Phosphoinositide Organization by Protein Electrostatics. Nature 2005, 438, 605–611.


Page 22 of 27

Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24)

Yeung, T.; Gilbert, G. E.; Shi, J.; Silvius, J.; Kapus, A.; Grinstein, S. Membrane Phosphatidylserine Regulates Surface Charge and Protein Localization. Science 2008, 319, 210–213.

(25)

Gambhir, A.; Hangyás-Mihályné, G.; Zaitseva, I.; Cafiso, D. S.; Wang, J.; Murray, D.; Pentyala, S. N.; Smith, S. O.; McLaughlin, S. Electrostatic Sequestration of PIP2 on Phospholipid Membranes by Basic/Aromatic Regions of Proteins. Biophys. J. 2004, 86, 2188–2207.

(26)

Samanta, S.; Hezaveh, S.; Roccatano, D. Theoretical Study of Binding and Permeation of Ether-Based Polymers through Interfaces. J. Phys. Chem. B 2013, 117, 14723–14731.

(27)

Schwieger, C.; Achilles, A.; Scholz, S.; Rüger, J.; Bacia, K.; Saalwaechter, K.; Kressler, J.; Blume, A. Binding of Amphiphilic and Triphilic Block Copolymers to Lipid Model Membranes: the Role of Perfluorinated Moieties. Soft Matter 2014, 10, 6147–6160.

(28)

Duan, X.; Ding, M.; Zhang, R.; Li, L.; Shi, T.; An, L.; Huang, O.; Xu, W. S. Effects of Chain Rigidity on the Adsorption of a Polyelectrolyte Chain on Mixed Lipid Monolayer: A Monte Carlo Study. J. Phys. Chem. B 2015, 119, 6041–6049.

(29)

Howes, P.; Green, M.; Levitt, J.; Suhling, K.; Hughes, M. Phospholipid Encapsulated Semiconducting Polymer Nanoparticles: Their Use in Cell Imaging and Protein Attachment. J. Am. Chem. Soc. 2010, 132, 3989–3996.

(30)

Cui, Q.; He, F.; Wang, X.; Xia, B.; Li, L. Gold Nanoflower@Gelatin Core−Shell Nanoparticles Loaded with Conjugated Polymer Applied for Cellular Imaging. ACS Appl. Mater. Interfaces 2013, 5, 213–219.

(31)

Yang, Y.; Wang, X.; Cui, Q.; Cao, Q.; Li, L. Self-Assembly of Fluorescent Organic Nanoparticles for Iron(III) Sensing and Cellular Imaging. ACS Appl. Mater. Interfaces 2016, 8, 7440–7448. 23 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32)

Page 24 of 27

Liu, B.; Bazan, G. C. Optimization of the Molecular Orbital Energies of Conjugated Polymers for Optical Amplification of Fluorescent Sensors. J. Am. Chem. Soc. 2006, 128, 1188–1196.

(33)

Li, X.; Guo, X.; Cao, L.; Xun, Z.; Wang, S.; Li, S.; Li, Y.; Yang, G. Water-Soluble Triarylboron Compound for ATP Imaging in Vivo Using Analyte-Induced Finite Aggregation. Angew. Chem. Int. Ed. 2014, 53, 7809–7813.

(34)

Hope, M. J.; Bally, M. B.; Webb. G.; Cullis, P. R. Production of Large Unilamellar Vesicles by a Rapid Extrusion Procedure. Characterization of Size Distribution, Trapped Volume and Ability to Maintain a Membrane Potential. Biochim. Biophys. Acta 1985, 812, 55–65.

(35)

Kahveci, Z.; Martínez-Tomé, M. J.; Mallavia, R.; Mateo, C. R. Use of the Conjugated Polyelectrolyte Poly{[9,9-bis(6’‑N,N,N‑trimethylammonium)hexyl]fluorene-phenylene}

Bromide

(HTMA-PFP) as a Fluorescent Membrane Marker. Biomacromolecules 2013, 14, 1990–1998. (36)

Xu, Z.; Singh, N. J.; Lim, J.; Pan, J.; Kim, H. N.; Park, S.; Kim, K. S.; Yoon, J. Unique Sandwich Stacking of Pyrene-Adenine-Pyrene for Selective and Ratiometric Fluorescent Sensing of ATP at Physiological pH. J. Am. Chem. Soc. 2009, 131, 15528–15533.

(37)

Xu, Z.; Kim, S. K.; Yoon, J. Revisit to imidazolium receptors for the recognition of anions: highlighted research during 2006–2009. Chem. Soc. Rev. 2010, 39, 1457-1466.

(38)

Feng, L.; Liu, L.; Lv, F.; Bazan, G. C.; Wang, S. Preparation and Biofunctionalization of Multicolor Conjugated Polymer Nanoparticles for Imaging and Detection of Tumor Cells. Adv. Mater. 2014, 26, 3926–3930.


Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(39)

Jiang, R.; Lu, X.; Yang, M.; Deng, W.; Fan, Q.; Huang, W. Monodispersed Brush-Like Conjugated Polyelectrolyte Nanoparticles with Efficient and Visualized SiRNA Delivery for Gene Silencing. Biomacromolecules 2013, 14, 3643–3652.

(40)

Shi, H.; Sun, H.; Yang, H.; Liu, S.; Jenkins, G.; Feng, W.; Li, F.; Zhao, Q.; Liu, B.; Huang, W. Cationic Polyfluorenes with Phosphorescent Iridium(III) Complexes for Time-Resolved Luminescent Biosensing and Fluorescence Lifetime Imaging. Adv. Funct. Mater. 2013, 23, 3268–3276.

(41)

Zhao, Q.; Zhou, X.; Cao, T.; Zhang, K. Y.; Yang, L.; Liu, S.; Liang, H.; Yang, H.; Li, F.; Huang, W. Fluorescent/Phosphorescent Dual-Emissive Conjugated Polymer Dots for Hypoxia Bioimaging. Chem. Sci. 2015, 6, 1825–1831.

(42)

Zhao, Q.; Zhang, C.; Liu, S.; Liu, Y.; Zhang, K. Y.; Zhou, X.; Jiang, J.; Xu, W.; Yang, T.; Huang, W. Dual-Emissive Polymer Dots for Rapid Detection of Fluoride in Pure Water and Biological Systems with Improved Reliability and Accuracy. Sci. Rep. 2015, 5, 16420–16430.

(43)

Förster, T. Intermolecular Energy Migration and Fluorescence. Ann. Phys. 1948, 2, 55-75.

(44)

Cingil, H. E.; Storm, I. M.; Yorulmaz, Y.; te Brake, D. W.; de Vries, R.; Stuart, M. A. C.; Sprakel, J. Monitoring Protein Capsid Assembly with a Conjugated Polymer Strain Sensor. J. Am. Chem. Soc. 2015, 137, 9800–9803.

(45)

Kummer, S.; Knoll, A.; Socher, E.; Bethge, L.; Herrmann, A.; Seitz, O. PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells. Bioconjugate Chem. 2012, 23, 2051–2060.

(46)

Wu, S.; Duan, N.; Ma, X.; Xia, Y.; Yu, Y.; Wang, Z.; Wang, H. Simultaneous Detection of Enterovirus 71 and Coxsackievirus A16 using Dual-Colour Upconversion Luminescent Nanoparticles as Labels. Chem. Commun. 2012, 48, 4866–4868.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(47)

Fan, G.; Lin, Y.; Yang, L.; Gao, F.; Zhao, Y.; Qiao, Z.; Zhao, Q.; Fan, Y.; Chen, Z.; Wang, H. Co-Self-Assembled Nanoaggregates of BODIPY Amphiphiles for Dual Colour Imaging of Live Cells. Chem. Commun. 2015, 51, 12447–12450.

(48)

Lim, C. S.; Kim, E. S.; Kim, J. Y.; Hong, S. T.; Chun, H. J.; Kang, D. E.; Cho, B. R. Measurement of the Nucleus Area and Nucleus/Cytoplasm and Mitochondria/Nucleus Ratios in Human Colon Tissues by Dual-Colour Two-Photon Microscopy Imaging. Sci. Rep. 2015, 5, 18521–18531.


Page 26 of 27

Page 27 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents


Binding-Directed Energy Transfer of Conjugated Polymer Materials for

Recommend Documents