Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

Jeppe Madsen,Nicholas J Warren,Russell Pearson,Irene Canton,Xiaohe Tian,Steven P Armes,Giuseppe Battaglia,Adam Blanazs,Efrosyni Themistou,Andrew L Lewis,Burcin Ustbas

doi:10.1021/ja407380t

Abstract

Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance.

Highlights

Fluorescent probes are widely used for imaging in cell biology
Deprotonation of (a) NB, (b) Nile Blue methacrylamide (NBM), and (c) NBCa aThe monocationic form of Nile Blue is stabilized by resonance, thereby increasing its pKa to around 10
(methacryloyloxy)ethyl carbamate (NBC) monomers used in this work is summarized in Scheme 1

Summary

■ INTRODUCTION

Fluorescent probes are widely used for imaging in cell biology. Increasingly, they are being utilized as chemosensors to diagnose specific pathological conditions and report on cellular events.[1−3] There is considerable interest in designing nanoparticles that report on physiologically relevant species such as ions,[4−6] reactive oxygen species,[7−9] gaseous biological second messengers[10−12] and hydrogen ions.[9,13,14] In particular, pH probes have diagnostic potential because many diseases are associated with changes in the local pH.[15]. The copolymer chains acquire cationic character and in principle may bind electrostatically to negatively charged cell membranes, internalization does not occur unless the local concentration of cationic charge density becomes high enough to disrupt the membrane.[59,60] The relatively small size of the copolymer chains[61] makes endocytosis unlikely, and this process is further hindered by the acidic environment.[62] these new biocompatible Nile Blue-based copolymer probes enable the differential interstitial pH of live spheroids to be monitored continuously and noninvasively in real time, as opposed to the indirect approach required for fixed tissue based on Glut-1 This is important because, the interstitial pH decreases, the intracellular pH remains at physiological levels in tumoral masses.[25,26,63] Importantly, no differential staining was observed when the MCTS model was treated with PMPC25−PDPA61-NB0.13 (Figure 2). This indicates the uniform localization of this copolymer, as previously observed with rhodamine-labeled copolymers.[36]

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES