Efficient cytosolic delivery of luminescent lanthanide bioprobes in live cells for two-photon microscopy

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.