Stability and restructuring of oleyl-modified hyaluronan micelles: effect of hydrophobic compounds and comparative evaluation of CMC determination methods

Amphiphilic hyaluronic acid conjugates form micellar nanostructures with critical micelle concentration (CMC) values that vary widely across studies due to differences in analytical methodology and experimental conditions. In particular, probe-based fluorescence approaches may influence micelle stability, thereby complicating direct comparison of published data. In this work, we investigated the self-assembly of oleate-modified hyaluronic acid (HA-C18:1) micelles using spectrofluorimetry (CMC = 0.071 ± 0.005 mg/mL), pendant drop surface tension (CMC = 0.140 ± 0.012 mg/mL), dynamic light scattering, and isothermal titration calorimetry (ITC; CMC = 0.025 ± 0.007 mg/mL). ITC provided a probe-independent thermodynamic assessment, revealing the lowest, most consistent CMC for primary micellization and highlighting method-dependent variability in conventional approaches. Encapsulation of hydrophobic guests—paclitaxel (PTX), carbobenzoxy-etoposide (ETP-Cbz), and oleic acid-stabilized iron oxide nanoparticles—stabilized the micelles and further reduced the apparent CMC. Notably, PTX-loaded micelles exhibited rod-like morphology and the lowest CMC (0.006 mg/mL), contrasting with spherical ETP-Cbz micelles (0.025 mg/mL), indicating guest-induced restructuring and enhanced stability. Overall, the results provide a comparative thermodynamic analysis of HA-based amphiphilic systems and highlight the influence of analytical methodology and guest molecules on micellar stability.