Analyzing Spatial Variations in Molecular Mobility in Hydrated Amorphous Drug-Polymer Blends Using Fourier Transform Fluorescence Recovery After Photobleaching and Image Segmentation

Amorphous solid dispersions (ASDs) can enhance oral bioavailability of poorly soluble drugs but often undergo hydration-induced phase separation, which complicates drug release and undermines physical stability. Here we integrate diffractive-optics–enabled Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with image segmentation in a controlled “glass-sandwich” hydration platform to quantify phase-resolved molecular mobility within hydrating ibuprofen–copovidone compacts. Release studies distinguished a rapidly releasing 10% w/w drug loading (DL) ibuprofen ASD from a poorly releasing 15% DL ASD. Confocal fluorescence microscopy revealed discrete, dispersed hydrophobic domains following hydration at 10% DL versus a persistent, connected drug-rich barrier that formed at the ASD-buffer interface for the 15% DL ASD. Dynamic light scattering studies of solutions following release from 10% DL ASD compacts demonstrated the presence of nanodroplets of 520 ± 8 nm, consistent with observations from microscopy studies. FT-FRAP with segmentation (rhodamine 6G probe) resolved domain-specific transport kinetics through elucidation of regional values of the probe diffusivity ( D ): low-hydration ASD region ( D trans < 0.017 μm 2 ·s –1; D rot = 0.21 ± 0.01 radians 2 ·s –1 ), ASD gel ( D trans = 0.91 ± 0.07 μm 2 ·s –1 ), aqueous regions ( D trans > 120 μm 2 ·s –1 ), and amorphous ibuprofen-rich phase ( D trans = 0.082 ± 0.002 μm 2 ·s –1 ). Stokes–Einstein estimates translated these values into local viscosities spanning ∼0.41 Pa·s (gel), 4.5 Pa·s (ibuprofen-rich), and 22 Pa·s (low-hydration ASD), highlighting pronounced microheterogeneity within the hydrated ASD matrix. Together, these results provide a quantitative, spatially explicit link between drug loading, phase morphology, local mobility, and macroscopic release, and establish segmented FT-FRAP as a practical tool for diagnosing failure mechanisms and guiding formulation of high-loading ASDs.