Carbohydrate-Mediated Cellular Uptake of Boronic Acid Hybrids In Cellulo and Insulin-Deficient Zebrafish: Live Imaging and Application of Multiphoton FLIM.

Understanding how synthetic molecular probes interact with biological carbohydrates is essential for theranostics design and discovery. We report new supramolecular polysaccharide complexes, comprising simultaneously functionalised boronic acids conjugated to xanthate-based fluorescent tags bound to saccharides such as glucose or β-D-glucan (a natural product from Hordeum Vulgare). These hybrids were investigated in solution and in the cellular milieu using advanced biophysical methods, including multiphoton fluorescence lifetime imaging microscopy (MP-FLIM). A new type of fluorescent hybrid nanoparticle was also generated by conjugating boronic acids to ─Si(OH)2 functionalities on the surface of core-shell Fe3O4@SiO2 nanoparticles. Cellular uptake and distribution were probed using correlated confocal and multiphoton fluorescence microscopy using 2-photon excitation at 910 nm, whereby TCSPC and FLIM provided insight into probe environments, interactions with β-D-glucan, and cellular context. Colocalization studies with a range of standard dyes were carried out to assess the potential of the probes for cellular penetration and targeting cellular organelles, and the nature of the lipophilic linker involved greatly influences the cytoplasmic distribution and facilitates zebrafish uptake. Complementary in vivo investigations using fluorescence stereomicroscopy of zebrafish showed differential uptake and quenching of these complexes throughout the digestive system, dependent on exogenous and endogenous concentrations of carbohydrates, glucose, or β-d-glucan.