Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

https://doi.org/10.1016/j.jphotobiol.2017.03.017Get rights and content

Highlights

  • Phenylenevinylene COEs were tested as cell membrane fluorescent markers.

  • PV-COEs were found to stain mammalian cell membrane-rich organelles.

  • Tested PV-COEs showed negligible toxicity towards cancerous and non-cancerous cells.

  • Distyrylstillbene derivative proved to be the best candidate for cell staining.

Abstract

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining was determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.

Introduction

Fluorescent staining is one of the most developing areas in biological and biomedical sciences. Over the last few years, great efforts have been developed to prepare highly specific fluorescent molecules for cellular studies and life cell imaging [1], [2], [3], [4], [5], [6], [7]. To meet the requirement of good optical properties, such as high quantum yield, excitation and emission range, excellent photostability, biocompatibility and low cytotoxicity, a promising approach seems to be employment conjugated polyelectrolytes (CPEs) as well as their shorter and well defined oligomeric counterparts, conjugated oligoelectrolytes (COEs), for this purpose [8], [9]. COEs belongs to a class of specialized molecules with π-conjugated molecular frameworks (which provide active optical properties) equipped with pendant groups bearing ionic functionality (which provide solubility in polar media) [10]. Their molecular architecture allow for interaction or formation of complexes with positively or negatively charged biomacromolecules and supramolecular structures, such as DNA, lipid membranes etc. [11], [12], [13], [14], [15].

Due to the unique properties, conjugated oligo- and polyelectrolytes have been involved in various biological studies including those aimed at visualization or detection of specific peptides and proteins [16] [17], cellular imaging [18], [19], and drug tracking and gene delivery systems [7], [14], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29]. As opposed to the immunostaining approach [30], [31], these examples highlight the applicability of CPEs and/or COEs as versatile and functional optical markers for human cell imaging and analysis. The unique properties of CPEs and COEs, specifically their optical activity and combination of hydrophobic and hydrophilic components, are anticipated to continue to afford exciting opportunities to develop functional materials and optical markers that utilize and exploit intermolecular or electrostatic interactions to expand the scope and capability of bioimaging and medical techniques.

The merger of materials chemistry, biology and medicinal sciences that has resulted in the current state-of-the-art of functional COE/CPE markers has the potential to meet the demands and challenges of imaging, diagnosis and therapy. However, most of the applications using conjugated oligo- and polyelectrolytes are based on complexes between CPEs or COEs with specific compounds that facilitate selective staining or other target applications. In this research a series of structurally related conjugated oligo(phenylenevinylene) derivatives were investigated for use as fluorescent stains for cellular imaging. The compounds discussed here allow visualization of membranous-based cellular compartments without the need of complexation partners. Chemically, all of the tested COEs contain a hydrophobic backbone, composed of π-delocalized phenylenevinylene repeat units and ammonium or carboxylate terminated pendant groups bound at both extremities. In our study we used the selected positively charged three- (distyrylbenzene-DSBN+), four- (distyrylstillbene-DSSN+) and five-ringed (COE1-5C+) COEs with ammonium ionic groups and one negatively charged four-ring analog (COE1-4C) with carboxylate ionic groups in place of cationic trimethylammonium pendant group terminals (Fig. 1).

The COE compounds used in this study exhibit unique photophysical and electronic properties coupled with solubility in the aqueous environments [32], [33] commonly inherent to sensing in biological systems and bioassays of proteins and nucleic acids [34], [35]. Phenylenevinylene COEs with this molecular structure are optically active and are known to undergo intra-molecular charge transfer excitation, which makes them sensitive to the polarity of the environment [36], [37], [38]. This class of COEs has been extensively studied in terms of linear and non-linear optical features, self-assembly properties and as charge transfer mediators in microbial fuel cells (MCFs) [39], [40], [41] and also as components that enable electrode driven microbial metabolism [19]. PV-COEs have been also extensively studied for uses as artificial LHC (light harvesting complexes) [42] and as a photocurrent generator in solar cells [43].

The fluorescence emission of the COEs employed in this study is higher when localized inside cellular lipid bilayers compared to when dissolved in polar media. This results from increased fluorescence quantum efficiency in non-polar surroundings, such as lipid membranes, relative to polar [19], [33]. It was demonstrated that DSSN+ and DSBN+ could be incorporated into both synthetic and natural lipid bilayer membranes in ordered molecular orientations [33]. Emission intensity profiles of stationary multi-lamellar phospholipid vesicles (with molar ratios of COE/lipid < 0.03) obtained following excitation with a linearly polarized source indicated that the hydrophobic core of the COEs positions within the inner-membrane region oriented with long molecular axes perpendicular to the plane of the lipid bilayers suggests these compounds might be suitable candidates for cellular membrane fluorescent probes [33]. The affinity of PV COEs for bacteria [40], [44] and yeast [19], [33] membranes has been demonstrated previously. It should be noted that one of these dyes (DSSN+) has already been used for extracellular vesicle (exosome) staining [45]. Initial attempts to employ PV COEs for mammalian cell culture staining have also been reported [19], [33], [46]. The results of these studies prompted us to investigate the applicability of such compounds towards a broad range of cells; specifically we aimed to probe their membrane staining features, specific localization within eukaryotic cells, and potential use as markers for fluorescence activated cell sorting (FACS) analysis [27], [47]. We have applied the COEs as staining agents for bioimaging of different types of human (including primary human cells) and mouse cells.

Section snippets

Synthesis of Conjugated Oligoelectrolytes

The three-ring positively charged, 1,4-bis(4′-(N,N-bis(6″(N,N,N-trimethylammonium)hexyl)amino) styryl) benzene tetraiodide (DSBN+), was synthesized as previously reported [32], [33], [38]. The four-ring positively charged, 4,4′-bis(4′-(N,N-bis(6″-(N,N,N-trimethylammonium)hexyl)amino)-styryl) stilbene tetraiodide (DSSN+), has been prepared as previously reported [33] with the modifications described in supplementary materials. The longer five-ring positive analog, 1,4-bis(4′-(N,N-bis(6″-(N,N,N

Motivation for Employment of Each PV COE for Cellular Staining

We synthetized and characterized a series of water-soluble phenylenevinylene COEs of various lengths and bearing cationic or anionic polar functional groups (Figs. 1 and S1). The synthesis of the three-ring distyrylbenzene (DSBN+) and four-ring distyrylstilbene (DSSN+) analogues has been reported previously [32], [33]. However, in the current work, the procedure was modified as described in Supplementary Materials. The three-ring and four-ring cationic COEs have been shown to have high affinity

Conclusions

In this study four phenylenevinylene-conjugated oligoelectrolytes were examined as a potential membrane staining dyes for mammalian cell imaging. These COEs all possess a particular molecular architecture in which a hydrophobic backbone with delocalized π electrons is functionalized at both extremities with pendant groups bearing terminal ammonium or carboxylate functionality. Three of the tested COEs, DSBN+, DSSN+, COE1-5C+ contain positive charged ammonium groups while COE1-4C (a negatively

Acknowledgements

We are thankful Jan Dhont, Peter Lang and Marc Obiols-Rabasa from the ESMI program. This work was supported by National Science Centre of Poland (NCN-2012/05/B/ST5/00364) and state funds for Polish Academy of Sciences. Confocal microscope analysis was performed at The Physical Chemistry Department, Lund University under the European Society for Molecular Imaging (ESMI) program.

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