D-(+)-Biotinylated squaraine dyes: A journey from synthetic conception, photophysical and -chemical characterization, to the exploration of their photoantitumoral action mechanisms

Biotin is primarily taken up by cells through sodium-dependent multivitamin transporter, which is highly expressed in aggressive cancer cell lines, often at levels surpassing those of the folate receptor. This makes biotin an attractive ligand for tumor-targeted drug delivery. Building on this rationale, this study presents a series of six D-(+)-biotin-conjugated squaraine dyes derived from benzothiazole, indolenine, and benz[e]indole, with N-ethyl and N-hexyl chains. These compounds were thoroughly characterized in terms of their photophysical and photochemical properties, revealing strong absorption in the so-called “phototherapeutic window”, notable fluorescence, especially the benzothiazole derivatives, aqueous stability, particularly the indolenine-based dyes, and moderate to high photostability. Computational studies further indicated a strong binding affinity to human serum albumin and avidin proteins. All dyes exhibited photodynamic activity, with indolenine derivatives showing remarkable tumor selectivity and benz[e]indole analogs evidencing superior photocytotoxicity. The most promising compounds preferentially accumulated in mitochondria, and both singlet oxygen and other reactive oxygen species were found to play a role in their photobiological effects. Additionally, they were non-genotoxic in the absence of irradiation, and apoptosis was the primary mechanism of cell death upon light activation. This was evidenced by preserved cytoplasmic membrane integrity, nuclear fragmentation, and caspase-3/7 activation, reinforcing the safety and potential of these compounds as phototherapeutic agents. Although cellular uptake via the sodium-dependent multivitamin transporter was not established, and diffusion is expected to be the predominant mechanism, the high predicted avidin-binding affinity of these dyes opens exciting new avenues for photodynamic therapy-combined strategies.