Development, characterization and application of nanoprobes for advanced FLIM studies of synthetic and cellular hydrogels

An abnormally high viscosity of the hydrogel layer on the airway epithelium is the characteristic hallmark of many respiratory tract diseases. Existing knowledge relies on measurement of bulk samples. However, little is known about the regional viscosity of mucus on living cells and organs, even less is known about its spatial distribution.

In the first funding period, we analyzed the fluorescence lifetime signatures of fluorescent molecular rotor (FMR) dyes on mucus and mucus-mimicking hydrogels. With Cy3 as viscosity-sensitive nanoprobe we established a fluorescence lifetime imaging microscopy (FLIM)-based protocol for measurements of viscoelastic properties of mucus and mucus-mimicking hydrogels in small samples. When applied to sputum samples from human donors, a remarkable heterogeneity of FMR-derived viscosity was observed. Increased viscosity of mucus from CF patients was also detected on air-liquid-interface (ALI) cell cultures. In this work, lectins were used to bring the FMR dye to the cell surface glycans. On the chemical side of the project, we pursued the development of small molecule probes, to facilitate high resolution imaging. In order to introduce the smallest possible cargo, we have optimised a method so that the covalent labelling of sialic acids on the surfaces of living cells can be achieved at 25 °C in a total time of only 15 minutes. Conjugates of FMR dyes with cell impermeable peptide hydrazides enabled fluorogenic cell surface staining. Ratio imaging after co-labelling with an environmentally insensitive dye showed that the glycan bound FMR responds to changes induced by the mucolytic agent TCEP. We systematically analysed cell surface staining with boronylated peptide conjugates. We identified “boronolectin” probes binding the cell surface with K_d = 0.9 µM. Interestingly, the fluorescence lifetime of FMR conjugates depends to a small but detectable extent on the bound sugar, suggesting that decoupling the viscosity sensor from the sugar sensor is a challenge.