Protocol-Dependent Effects on Colloidal Characterization and Drug Loading/Release Analysis of Thermosensitive PNIPAM-co-COOH Microgels

This work analyzes protocol-dependent effects on the colloidal characterization and drug loading/release analysis of model thermosensitive PNIPAM-co-COOH microgels and shows how they can be quantified or minimized through targeted methodological refinements. Findings reveal that standard single-beam DLS underestimates the collapsed hydrodynamic radius by 18% at 43 °C due to thermal convection. After drift correction, 3D-DLS combined with SLS provides a consistent description of thermally induced collapse, pH-dependent swelling and core–corona structure. Regarding drug delivery, loading efficiency for Doxorubicin and 5-Fluorouracil is maximized near the volume phase transition temperature, where hydrophobic interactions are strongest. For release studies, dialysis is recommended, but free-drug blanks are required to account for membrane-induced delay and ensure accurate early kinetic profiles. By integrating TEM, AFM, SLS, DLS, NTA and LDE, this study establishes a robust framework for the colloidal characterization of thermosensitive microgels. These refinements reduce experimental bias and may be extended to related soft nanocarriers.