Synthesis and Characterisation of Bioactive Fluorescent FITC-Insulin Glulisine Conjugates for Potential Use in Insulin Delivery

Background/Objectives: Drug development and delivery remain critical areas of research for addressing modern bioanalytical challenges. Understanding drug biodistribution, stability, and metabolism within biological systems is essential for optimising therapeutic efficacy. This study focuses on synthesising and characterising a novel fluorescent conjugate derived from commercially available rapid-acting insulin glulisine (Apidra®) and fluorescein isothiocyanate (FITC). The objective was to produce a mono-labelled FITC-insulin glulisine conjugate without employing complex protective group strategies or multi-step processes. Methods: The conjugation was optimised by varying molar ratios (1:1 to 3:1) and reaction times (18–24 h) at pH 7. Results: The desired B1 mono-labelled conjugate was successfully achieved at a 2:1 molar ratio, pH 7, and 18 h reaction time. MALDI-TOF mass spectrometry confirmed the molecular weight and conjugation site, with fragmentation analysis identifying FITC attachment at phenylalanine (B1) on the β-chain (m/z = 537.11). Western blots performed on C2C12 skeletal cell lysates stimulated with the FITC–insulin glulisine conjugate showed Akt and IRS-1 activity similar to that of cells treated with native commercial insulin glulisine. Confocal imaging also demonstrated translocation of GLUT4 in FITC–insulin glulisine conjugate-treated C2C12 cells similar to that of commercial native insulin glulisine. Octanol-water partitioning studies assessed the physicochemical properties of the conjugate. Conclusions: This approach demonstrates an efficient method for fluorescent labelling of insulin analogues, enabling future applications in imaging, biodistribution studies, and pharmacokinetic profiling.