Enhanced NLCs: Elevating therapeutic and diagnostic performance through covalent dye integration

Nanostructured lipid carriers (NLCs) are widely investigated as versatile drug delivery systems. In this study, we aimed to design, synthesize, and characterize a novel fluorescently labeled NLC platform incorporating a rhodamine B–oleic acid conjugate (Rd-OA) as a proof-of-concept model for future therapeutic applications. NLCs were prepared using Precirol® ATO 5, oleic acid and Rd-OA, and Tween® 20 (T20) or Tween® 80 (T80). The resulting nanoparticles exhibited sizes of 350–500 nm, narrow polydispersity indices (<0.2), and highly negative zeta potentials (∼–30 mV), confirming stable colloidal properties. Rd-OA was efficiently integrated (100% incorporation efficiency) with different concentrations. Molecular dynamics simulations provided atomic-level structural insights, revealing surfactant-dependent differences in component organization consistent with experimental data. NLCs were stable over a period of three months. In vitro assays in keratinocytes cells demonstrated a concentration-dependent cytotoxicity effect influenced by Rd-OA content, while fluorescence microscopy confirmed efficient cellular internalization of the NLCs. Collectively, these findings support the developed fluorescent NLC system as a stable, reproducible, and traceable platform suitable for future adaptation in drug delivery and imaging applications.” • Developed a novel nanostructured lipid carrier (NLC) system covalently linked to a fluorescent dye for combined drug delivery and bioimaging. • Demonstrated enhanced nanoparticle stability, supported by in silico modeling predictions and experimental validation. • Achieved reliable real-time tracking and precise localization through covalent dye integration, minimizing dye leakage and false signals. • Validated the biocompatibility of the NLC system, ensuring safety for biomedical applications. • Presented a versatile, multifunctional nanocarrier platform with strong potential for clinical translation in theranostics and precision medicine.