Real-time fluorescence imaging of anthocyanins complexed with diphenylboric acid 2-aminoethyl inside B16-F10 melanoma cells

Abstract

Anthocyanins (AN), natural compounds daily consumed by humans, have outstanding therapeutical potential if administered topically in melanoma pathology. However, the search for efficient therapy development is still in progress, owing to the lack of complete understanding of the AN intracellular path, once they are uptaken by the cells. This target is constrained by the need for an imaging strategy that would enable their intracellular detection and localization in-situ. In this light, diphenylboric acid 2-aminoethyl (DPBA), a non-fluorescent reagent, was here successfully used to form fluorescent complexes with AN. The AN used are the cyanidin aglycon as a free standard molecule (CY), and the glycosylated compounds, extracted and purified from chokeberry fruits (AE). In solution, it was observed that the fluorescence emission increased by 39% (CY@DPBA), and by 34% (AE@DPBA), which concludes that AN form fluorescent complexes with DPBA (CY@DPBA and AE@DPBA). In addition, using NMR (nuclear magnetic resonance) spectroscopy, and HRMS (high-resolution mass spectrometry) analysis, the structure of the CY@DPBA complex was efficiently elucidated. In-vitro experiments showed that the complexes formed after the treatment proved to be non-toxic on B16-F10 cells. The sub-cellular visualization of all AN was monitored by fluorescence microscopy and flow cytometry, demonstrating detectable signals of the non-metabolized CY and glycosylated CY inside melanoma cells. This study reports that the use of DPBA to image AN intracellularly is a sensitive, non-invasive and successful method that can extend its application in broad fields like drug development or metabolism-associated mechanisms.

Keywords: 2-((diphenylboraneyl)oxy)ethan-1-amine; Anthocyanins; Fluorescence microscopy; HRMS; Melanoma B16–F10 cells; NMR.