NIR squaraine dyes for cell bilayer bioimaging: a structure-activity investigation

Near-infrared (NIR) emissive probes have recently attracted an increasing interest as stain of cell bilayer membranes.1 Membrane fluidity, polarization, as well as the specific interactions occurring among phospholipids, proteins and channels, are important to shed light on crucial biochemical pathways. In this context, small organic fluorescent compounds play a key role, allowing an accurate location within the membrane along with the possibility to fine-tune their optical features by simple structural modifications.2 In particular, NIR probes exhibit remarkable brightness, deep tissue penetration and limited biological damage and autofluorescence.3 Although several emitters have already been proposed for this purpose, a regular innovation in design of more performing probes is required.4 A systematic structure-to-function study for novel bilayer membrane NIR probes could represent the starting point to precisely direct further investigations. In this work, novel asymmetric squaraines have been synthesized and characterized. A carboxylic group linked to an indolenine (or benzoindolenine) ring has been placed on one side, while on the opposite one a perimidine derivative has been decorated with alkyl chains of different length. The former improves the water solubility of the whole system, while the latter tunes the interaction of the probe with the lipophilic components of the cell membrane. The kinetic of the probes’ intercalation inside the bilayers has been monitored by fluorescence spectroscopy. The effect of the membrane phase and fluidity on the interaction has been evaluated too, providing a complete analysis about the key structural motifs for the design of novel NIR probes.