Fluorescent Labeling of Inulin-Based siRNA Delivery Nanocarriers: Implications for Stability and Biological Performance.

Fluorescently labeled nanoparticles are often implicitly assumed to mimic the behavior of their unlabeled counterparts, despite potential physicochemical perturbations induced by probe conjugation. Herein, we systematically investigated the effect of fluorophore chemistry and labeling density on the colloidal stability and short interfering RNA (siRNA)-binding performance of inulin-based polymeric nanoparticles functionalized with branched polyethylenimine (bPEI) and poly-(D,l-lactic acid) (PLA). Two fluorophores, cyanine 7.5 (Cy7.5) and fluorescein isothiocyanate (FITC), were introduced via distinct conjugation strategies at variable grafting densities. Light scattering analyses revealed probe- and density-dependent modulation of nanoparticle size distribution and surface charge, with high FITC density inducing increased heterogeneity. Polyanion competition and RNase protection assays showed preserved siRNA stability for Cy7.5-labeled nanoparticles, whereas FITC conjugation reduced the level of siRNA retention at high labeling densities. Cellular uptake studies in MC38 cells demonstrated a clear overlap of fluorescent signals from Cy7.5 nanosystems and delivered siRNA under serum-free conditions, while serum proteins promoted partial siRNA displacement. Overall, these results demonstrate that fluorescent labeling is not a neutral modification and must be critically validated to avoid misinterpretation in fluorescence-based nanomedicine studies.