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Abstract

This study focuses on creating enhanced folate-conjugated fluorescent probes designed to target folate receptors, such as FRα, which are overexpressed in various cancers, including ovarian, breast, and lung. Such probes play a crucial role in fluorescence-guided surgery, a technique that enhances tumor visibility during operations. By improving tumor visualization, surgeons can more accurately remove cancerous tissue while preserving healthy structures, thereby improving cancer treatment outcomes. In the first probe type, the dye fluorescein was coupled to folate using an O-aminoserine linker for a facile oxime click reaction. Next, we synthesized probes with a disulfide linker that respond to elevated glutathione levels in cancer cells by disulfide cleavage, facilitating targeted fluorophore release and resulting in notable fluorescence enhancement within the cells. Finally, we prepared probes with very short linkers via the direct coupling of folate to 5- and 6-aminofluorescein. The photophysical properties and photostability of these probes were thoroughly evaluated. Cellular experiments demonstrated cancer-specific targeting, with a very high fluorescence intensity observed in cancerous (HeLa) cells compared to very low fluorescence in non-cancerous HDFa cells. Notably, the direct coupling of folic acid to 5-aminofluorescein resulted in a probe that displays particularly strong quenching in biological media if not bound to cancer cells, a desirable feature for reducing the signal-to-noise ratio in imaging applications. Furthermore, confocal microscopy demonstrated the effectiveness of the synthesized probes, achieving high-quality visualization of cancer cells and highlighting their potential for further preclinical evaluation in fluorescence-guided surgery.