Supramolecular Ensembles Formed between Charged Conjugated

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Supramolecular Ensembles Formed between Charged Conjugated Polymers and Glycoprobes for the Fluorogenic Recognition of Receptor Proteins Wei-Tao Dou, Ya-Li Zeng, Ying Lv, Jiatao Wu, Xiao-Peng He, Guo-Rong Chen, and Chunyan Tan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03223 • Publication Date (Web): 09 May 2016 Downloaded from http://pubs.acs.org on May 15, 2016

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ACS Applied Materials & Interfaces

Supramolecular Ensembles Formed between Charged Conjugated Polymers and Glycoprobes for the Fluorogenic Recognition of Receptor Proteins Wei-Tao Dou,1 Ya-Li Zeng,1 Ying Lv,2 Jiatao Wu,2 Xiao-Peng He,1* Guo-Rong Chen,1* and Chunyan Tan2,* 1

Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University of

Science and Technology, 130 Meilong Rd., Shanghai 200237, PR China 2

The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of

Chemical Biology, the Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China

ABSTRACT: This paper describes the simple construction of a unique class of supramolecular ensembles formed by the electrostatic self-assembly between charged conjugated polymers and fluorophore-coupled glycoligands (glycoprobes) for the selective, fluorogenic detection of receptor proteins at both the molecular and cellular levels. We show that positively and negatively charged diazobenzene-containing poly(p-phenylethynylenes) (PPEs) can be used to form stable fluorogenic probes with fluorescein (negatively charged) and rhodamine B (positively charged) based glycoprobes, respectively, by electrostatic interaction. The structure of the ensembles has been characterized by spectroscopic and microscopic techniques. The supramolecular probes 1

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formed show a quenched fluorescence in an aqueous buffer solution, which can be specifically recovered, in a concentration dependent manner, through the competitive complexation with a selective protein receptor, over a range of other unselective proteins. The ensembles also show a selective fluorescence enhancement with a live cell that expresses the glycoligand receptor, but not that without receptor expression.

Keywords: Conjugated polymer, probe, fluorescence, lectin, live cell, receptor

Introduction The selective recognition between receptor proteins, which exist on the membrane of mammalian cells, and their ligand molecules is responsible for a myriad of physiologically important processes. However, receptor-ligand interactions have also been associated with many diseases. For example, besides its physiological role to eliminate excessive asialoglycoproteins from the blood circulation, the asialoglycoprotein receptor (ASGPr) has also been identified as an invasion site of hepatotropic viruses and is overexpressed during liver inflammation.1,2 Macrophages feature two typical polarized phenotypes, which are the classically (M1) and alternatively (M2) activated macrophages. While the former can kill foreign organisms and cancer cells, the later that overexpresses on the cell membrane a mannose receptor facilitates tumorigenesis and cancer metastasis.3 As a result, glycoligand receptors have been viewed as promising biomarkers for disease diagnosis and targeted drug delivery.4-9 Owing to the typically low binding affinity between glycoligands and receptors, however, construction of potent tools for probing receptor-glycoligand recognitions is challenging.

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Here, we show the simple development of a unique class of supramolecular glyco-ensembles formed between charged conjugated polymers (CPs) and fluorophore-tagged glycoligands (glycoprobes) for the selective, fluorogenic detection of lectins (receptor proteins that recognize glycoligands) and live cells that express ASPGr. A number of decent strategies have been reported for the construction of multivalent glycopolymers to enhance the binding avidity with receptor proteins.10-18 Whereas these strategies generally require polymerization reactions, the supramolecular approach shown here simply depends on the electrostatic interaction between charged CPs and glycoprobes to acquire 1) a strong binding with receptors and 2) a fluorogenic sensing signal.

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Fig. 1. (a) Structure of the rhodamine B (RB) and fluorescein (Fluo) labeled glycoprobes (Gal, Glc and GalNAc are galactose, glucose and N-acetyl galactosamine, respectively) and alternating diazobenzenecontaining poly(p-phenylethynylene) (PPE1 and PPE2 are the positively and negatively charged PPEs, 3



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respectively) used in the study. (b) Schematic illustration of the supramolecular, electrostatic selfassembly between glycoprobes and PPEs and their use as fluorogenic probes for the detection of receptor-glycoligand recognitions. CPs are widely employed optical and electronic materials that strongly absorb light, and can be used as amplified quenchers for fluorophores.19-21 With these properties, CPs have proven to be suitable for the construction of fluorescence sensors.22-33 We have recently developed positively or negatively charged alternating diazobenzene-containing poly(p-phenylethynylene) (PPE), a unique class of CPs with an outstanding quenching ability for organic fluorescence dyes (Fig. 1a).34,35 Glycoligands coupled with either a rhodamine B (positively charged) or a fluorescein (negatively charged) dye are synthesized to form supramolecular ensembles with the PPEs by electrostatic interaction (Fig. 1b). The formed glyco-ensembles show a quenched fluorescence, which can be specifically recovered by, competitively, interaction with selective lectins, over a variety of other unselective proteins, in an aqueous buffer solution. The supramolecular probes also show a concentration-dependent fluorescence enhancement with a live cell that expresses ASGPr (a galactose receptor) but not a control cell without receptor expression. Experimental section

General. All purchased chemicals and reagents are of analytical grade. Proteins were purchased from Sigma-Aldrich. 1H NMR and 13C NMR spectra were recorded on a Bruker AM 400 MHz spectrometer with tetramethylsilane (TMS) as internal reference. Polymers were synthesized according to our previously published procedure.34,35 Absorption spectra were measured on a Varian Cary 500 UV-Vis spectraphotometer. High-Resolution Mass Spectra were recorded using a Waters LCT Premier XE spectrometer. High Performance Liquid Chromatography (HPLC) was performed on a Shimadzu Prominence Series equipment. 4


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Fluorescence spectroscopy. In a typical fluorescence quenching assay, the glycoprobes were incubated with a PPE of different concentrations in Tris-HCl (0.01 M, pH 7.4) for 30 s, and then the fluorescence was measured on a Varian Cary Eclipsefluorescence spectrophotometer with excitation of 510 nm and 450 nm for rhodamine B (RB) and fluorescein (Fluo), respectively. In a typical fluorescence assay for proteins, a protein solution of different concentrations was added to a mixed solution (0.01 M Tris-HCl, pH 7.4) of glycoprobe and PPE. Then, the resulting mixture was incubated at 25 °C for 10 min, and the fluorescence spectra were recorded at room temperature with excitation of 510 nm and 450 nm for RB and Fluo, respectively. In a typical fluorescence assay for cells, a cell line (Hep-G2 or HeLa, for cell-culture conditions in detail, see reference 40) of different concentrations was spotted to a microplate, and then a glycoprobe or a glycoprobe/PPE ensemble was added. After incubation for 15 min, the fluorescence of each well was determined by an M5 microplate reader (Molecular Device, USA) (excitation channel: 450 and 490 nm for RB and Fluo, respectively; emission channel: 510 and 580 nm for RB and Fluo, respectively) in a high throughput manner. Results and discussion

Monosaccharides including galactose (Gal), glucose (Glc) and N-acetyl galactosamine (GalNAc) were coupled with fluorophores by the Cu(I)-catalyzed azide-alkyne cycloaddition (click reaction) (Fig. 1a and Scheme S1).36,37 Fluorescein (negatively charge) and rhodamine B (positively charged) were employed to probe the generality of the strategy to form supramolecular glyco-ensembles. Both negatively charged PPE2 and positively charged PPE1 were produced by our previously established protocol.34,35

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Fig. 2. Fluorescence titration of (a) glyco-rhodamine B (1 µM, λex = 510 nm) with increasing PPE1 (080 µM for both Gal-RB and GalNAc-RB) and PPE2 (0-80 µM for both Gal-RB and GalNAc-RB), and (b) glyco-fluorescein (0.25 µM, λex = 450 nm) with increasing PPE1 (0-50 µM for both Gal-Fluo and Glc-Fluo) and PPE2 (0-60 µM for both Gal-Fluo and Glc-Fluo) in Tris-HCl (0.01 M, pH 7.4).

With the compounds in hand, the supramolecular, electrostatic self-assembly between glycoprobes and PPEs were carried out in an aqueous buffer solution.27,28,34 Fluorescence spectroscopy was first used to determine the association between the various glycoprobes and PPEs (Fig. 2). We observed concentration-dependent fluorescence quenching for all the glycoprobes in the presence of a PPE. Interestingly, while the fluorescence intensity of the positively charged Gal-RB and GalNAc-RB was quenched more significantly in the presence of the negatively charged PPE2 (Fig. 2a), that of the negatively charged Gal-Fluo and Glc-Fluo decreased more in the presence of the positively charged PPE1 (Fig. 2b). These observations, which are irrespective of the structure of 6


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the glycoligands attached (Gal, GalNAc or Glc), probably suggest that electrostatic contacts, in addition to other forces such as π-stacking, might be the main driving force to facilitate the binding between these dyes and PPEs. The double reciprocal plots used to determine the binding constants (k) between the glycoprobes and PPEs similarly suggest the stronger association between a glycoprobe and a PPE with the opposite charge (Fig. S1). Results shown in Fig. S2 indicated that with increasing NaCl concentration, the quenched fluorescence of the glycoprobe-PPE ensemble gradually recovered. This probably suggests a reversible Coulombic interaction between the two species, which can be compromised with the increase of salt concentration.

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We also used other techniques to characterize the assembly between PPEs and glycoprobes. Both scanning electron microscopy (SEM) (Fig. 3a) and dynamic light scattering (DLS) (Fig. 3b) suggest that the particle size of PPEs increased after assembly with a glycoprobe. Meanwhile, the zeta potential of Gal-RB/PPE2 increased and that of Gal-Fluo/PPE1 decreased with respect to PPE2 and PPE1 alone, respectively (Fig. 3c). These data suggest the electrostatic formation of the supramolecular glyco-ensembles.

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Fig. 4. Fluorescence titration of (a) Gal-RB/PPE2 (1 µM/80 µM, λex = 510 nm) and GalNAc-RB/PPE2 (1 µM/80 µM, λex = 510 nm) in the presence of increasing PNA (0-40 µM) and soybean agglutinin (SBA, 0-40 µM), respectively, and (b) Gal-Fluo/PPE1 (0.25 µM/50 µM, λex = 450 nm) and GlcFluo/PPE1 (0.25 µM/50 µM, λex = 450 nm) in the presence of increasing PNA (0-3 µM) and Con A (03 µM), respectively, in Tris-HCl (0.01 M, pH 7.4). Fluorescence change of (c) Gal-RB/PPE2 (1 µM/80 µM, λex = 510 nm, 40 µM protein) and GalNAc-RB/PPE2 (1 µM/80 µM, λex = 510 nm, 40 µM protein), and (d) Gal-Fluo/PPE1 (0.25 µM/50 µM, λex = 450 nm, 5 µM protein) and Glc-Fluo/PPE1 (0.25 8


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µM/50 µM, λex = 450 nm, 5 µM protein) in the presence of different proteins, where I and I0 are the fluorescence intensity of the supramolecular architecture with and without a protein, respectively, in TrisHCl (0.01 M, pH 7.4). (Abbreviations: PNA = Peanut agglutinin, SBA = Soybean agglutinin, Con A = Concanavalin A, Pep = Pepsin, BSA = Bovine serum albumin, WGA = Wheat germ agglutinin).

Next, we used lectin receptors that can recognize selectively glycoligands as a molecular model to test the sensing performance of the supramolecular ensembles in aqueous solution. The more strongly bound Gal-RB/PPE2, GalNAc-RB/PPE2, Gal-Fluo/PPE1 and Glc-Fluo/PPE1 were employed. Concentration-dependent fluorescence recovery of the probes in the presence of a selective lectin (i.e. Gal with Peanut agglutinin [PNA], Glc with Concanavalin A [Con A] and GalNAc with Soybean agglutinin [SBA]) was observed (Fig. 4a and 4b).4,38,39 The fluorescence increment showed good linearity over an increasing range of lectin concentrations, and the limit of detection (LOD) of the probes for the lectins were nanomole-ranged, with the glyco-Fluo/PPEs being the most sensitive (Fig. S3). To test the biospecificity of the probes, we used a panel of unselective proteins to incubate with the supramolecular ensembles. To our delight, the probes only showed a sharp fluorescence increase in the presence of the selective lectin, but minimal response to the unselective proteins (Fig. 4c and 4d, and Fig. S4). The “OFF-ON” fluorescence images of the glycoprobes with PPEs and lectins is shown in Fig. S5. To further test the sensitivity of the supramolecular ensembles for transmembrane receptors, a cellular assay was carried out. Human hepatoma cell line (Hep-G2) that expresses a galactose receptor (ASGPr)1,2,40 and human cervical cancer cell line (HeLa) without the receptor expression40 were employed. The live cells were spotted to a microplate, followed by the addition of a glycoprobe or an ensemble. After incubation for 15 min, we observed that the fluorescence of Gal-RB 9



and Gal-Fluo did not change (Fig. 5a). In contrast, the Gal-RB/PPE2 and Gal-Fluo/PPE1 with a largely suppressed background emission showed a gradually enhanced fluorescence with increasing Hep-G2, but not HeLa (Fig. 5b). This suggests that our ensembles could be also used to determine transmembrane receptors. The fact that a control cell concentration (HeLa) of up to 100,000 mL-1 caused a minimal change of their initial fluorescence (Fig. 5b) suggests the good stability of our CP-based electrostatic ensembles in a cellular medium. A subsequent cell viability assay indicated that the ensembles were not toxic to Hep-G2 as well as a healthy cell line (Lo2, human hepatic cell line) (Fig. 6).

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concentrations by an M5 microplate reader (Molecular Device, USA), where I0 and I are the fluorescence intensity of glycoprobe and that of the glycoprobe or ensemble with a cell line, respectively.

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Conclusion

We have shown the possibility of using PPEs as a polymeric backbone to construct fluorogenic supramolecular probes for detection of receptor proteins. The PPEs could spontaneously form unique supramolecular architectures with fluorophore-coupled glycoligands by electrostatic interaction, producing a quenched fluorescence signal. These ensembles showed specific fluorescence re-activation upon recognition of their selective receptor proteins in full aqueous solution. Additionally, the ensembles exhibited a concentration-dependent fluorescence recovery with a live cells that expresses a selective glycoligand receptor, but with minimal response to control cell 11



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without receptor expression. This study offers a new insight into the development of simple and effective supramolecular polymeric materials for biochemical applications based on CPs.

ASSOCIATED CONTENT Supporting Information Additional figures cited in this article, synthesis and characterization of new compounds and original spectral copy of new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] (X.-P. He) *Email: [email protected] (G.-R. Chen) *Email: [email protected] (C. Tan)

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT

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This research is supported by the 973 project (2013CB733700), the National Natural Science Foundation of China (21572058, 21576088, and 21572115) and the Shanghai Rising-Star Program (16QA1401400).

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Graphical Abstract

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Supramolecular Ensembles Formed between Charged Conjugated

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