Engineered ratiometric Sensory electrospun fibers for oxygen mapping in complex cultures and tumor microenvironment

Monitoring the hypoxic microenvironment is fundamental due to its implication in tumor aggressiveness and progression. In this work, we propose the fabrication of ratiometric fluorescent fibers via electrospinning of poly(trimethylsylil)propine (PTMSP) polymer, an optically clear and gas permeable polymer, for oxygen (O₂) sensing in a melanoma tumor model. The ratiometric sensing configuration was obtained by entrapping tris(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride, capable of detecting dissolved O₂ variations, together with rhodamine B isothiocyanate, serving as a reference dye, within the polymer matrix. The fibers were characterized to point out morphology, porosity, and hydrophilicity. The sensing ability of the fibrous mat was deeply investigated by means of microplate reader and confocal imaging, showing a strict correlation between the fluorescent ratiometric read-out and the increasing concentration of dissolved O₂ in aqueous-based media. Moreover, the fibers exhibited high photostability, reversibility and excellent cytocompatibility, allowing monitoring O₂ gradients over time and space in vitro melanoma co-cultures. Overall, the optimized micrometric sensing system holds potential for real-time assessments of dissolved O₂ levels in vitro complex cell systems and heterogeneous tumour microenvironments, and can open up new engineering possibilities by means of using O₂-sensitive dyes in tissue engineering scaffolding strategies.