Multiphoton fluorescence lifetime imaging microscopy (FLIM) and super-resolution fluorescence imaging with a supramolecular biopolymer for the controlled tagging of polysaccharides

Haobo Ge, Fernando Cortezon-Tamarit, Hui Chen Wang, Adam C. Sedgwick, Rory L. Arrowsmith, Vincenzo Mirabello, Stanley W. Botchway, Tony D. James, Sofia I. Pascu

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8 Citations (SciVal)


A new supramolecular polysaccharide complex, comprising a functionalised coumarin tag featuring a boronic acid and β-d-glucan (a natural product extract from barley, Hordeum Vulgare) was assembled based on the ability of the boronate motif to specifically recognise and bind to 1,2- or 1,3-diols in water. The complexation ratio of the fluorophore:biopolymer strand was determined from fluorescence titration experiments in aqueous environments and binding isotherms best described this interaction using a 2:1 model with estimated association constants of K2:1a1 = 5.0 × 10 4 M -1 and K2:1a2 = 3.3 × 10 11 M -1 . The resulting hybrid (denoted 5@β-d-glucan) was evaluated for its cellular uptake as an intact functional biopolymer and its distribution compared to that of the pinacol-protected coumarin boronic acid derivative using two-photon fluorescence lifetime imaging microscopy (FLIM) in living cells. The new fluorescent β-d-glucan conjugate has a high kinetic stability in aqueous environments with respect to the formation of the free boronic acid derivative compound 5 and retains fluorescence emissive properties both in solution and in living cells, as shown by two-photon fluorescence spectroscopy coupled with time-correlated single photon counting (TCSPC). Super-resolution fluorescence imaging using Airyscan detection as well as TM AFM and Raman spectroscopy investigations confirmed the formation of fluorescent and nano-dimensional aggregates of up to 20 nm dimensions which self-assemble on several different inert surfaces, such as borosilicate glass and mica surfaces, and these aggregates can also be observed within living cells with optical imaging techniques. The cytoplasmic distribution of the 5@β-d-glucan complex was demonstrated in several different cancer cell lines (HeLa and PC-3) as well as in healthy cells (J774.2 macrophages and FEK-4). Both new compounds (pinacol protected boronated coumarin) 5-P and its complex hybrid 5@β-d-glucan successfully penetrate cellular membranes with the minimum morphological alterations to cells and distribute evenly in the cytoplasm. The glucan biopolymer retains its activity towards macrophages in the presence of the coumarin tag functionality, demonstrating the potential of this natural β-d-glucan to act as a functional self-assembled theranostic scaffold capable of mediating the delivery of anchored small organic molecules with imaging and drug delivery applications.

Original languageEnglish
Pages (from-to)9498-9507
Number of pages10
Issue number19
Early online date1 Apr 2019
Publication statusPublished - 21 May 2019

ASJC Scopus subject areas

  • Materials Science(all)


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