ABSTRACT
Lanthanide(iii) (Ln3+) complexes have desirable photophysical properties for optical bioimaging. However, despite their advantages over organic dyes, their use for microscopy imaging is limited by the high-energy UV excitation they require and their poor ability to cross the cell membrane and reach the cytosol. Here we describe a novel family of lanthanide-based luminescent probes, termed dTAT[Ln·L], based on (i) a DOTA-like chelator with a picolinate moiety, (ii) a two-photon absorbing antenna to shift the excitation to the near infrared and (ii) a dimeric TAT cell-penetrating peptide for cytosolic delivery. Several Tb3+ and Eu3+ probes were prepared and characterized. Two-photon microscopy of live cells was attempted using a commercial microscope with the three probes showing the highest quantum yields (>0.15). A diffuse Ln3+ emission was detected in most cells, which is characteristic of cytosolic delivery of the Ln3+ complex. The cytotoxicity of these three probes was evaluated and the IC50 ranged from 7 µM to >50 µM. The addition of a single positive or negative charge to the antenna of the most cytotoxic compound was sufficient to lower significantly or suppress its toxicity under the conditions used for two-photon microscopy. Therefore, the design reported here provides excellent lanthanide-based probes for two-photon microscopy of living cells.
ABSTRACT
In the past, Lanthanide Luminescent Bioprobes (LLBs) based on pyclen-bearing π-extended picolinate antennas were synthesized and demonstrated well-adapted optical properties for biphotonic microscopy. The objective of this work is to develop a strategy to design bifunctional analogues of the previously studied LLBs presenting an additional reactive chemical group to allow their coupling to biological vectors to reach deep in vivo targeted two-photon bioimaging. Herein, we elaborated a synthetic scheme allowing the introduction of a primary amine on the para position of the macrocyclic pyridine unit. The photophysical and bioimaging studies demonstrate that the introduction of the reactive function does not alter the luminescent properties of the LLBs paving the way for further applications.
