Department of Chemistry, Royal College of Surgeons in Ireland (RCSI), D02 PN40 Dublin, Ireland.
Molecules. 2024 May 24;29(11):2474. doi: 10.3390/molecules29112474.
Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.
具有区分细胞内酸度亚细胞变化能力的分子探针对于研究动态细胞过程和功能仍然很重要。在这种情况下，已经开发了一系列具有最大发射波长分别为 720nm（在 pH > 6 时）和 790nm（在 pH < 5 时）的环状肽和 PEG 生物共轭双近红外发射 BF-偶氮二吡咯甲川荧光团，并确定了其在水溶液中的光物理性质。它们的互变发射和荧光寿命特性被用来跟踪它们从与质膜的首次接触到亚细胞区室再到在细胞外囊泡中释放的空间和时间进展。荧光团上的酚盐/酚的 pH 依赖性可逆互变控制了双发射响应的动态变化和相应的寿命变化。在转移性乳腺癌细胞系 MDA-MB-231 中的活细胞共焦显微镜实验证实了双发射性质在长时间内成像的可用性。所有三种衍生物都可作为探针，能够实时连续成像基本的细胞过程，如质膜相互作用、追踪内吞作用、溶酶体/大酸性囊泡积累和细胞外囊泡内的流出，而不会干扰细胞功能。此外，荧光寿命成像显微镜提供了关于荧光团随时间在细胞内微环境中进展的有价值的见解。总的来说，这些荧光团的独特光物理性质显示出它们作为信息丰富的探针的巨大潜力。
