Deep-Learning-Enabled SEM Image Segmentation Coupled with Laser Confocal Raman Microscopy: Elucidating Microstructure and Drug Spatial Distribution in Leuprorelin Acetate Microspheres

Background/Objectives: The precise characterization of the key microstructural and physicochemical attributes in sustained-release microspheres remains a technical bottleneck, hindering the understanding of drug release mechanisms, and limiting insights into the “process–structure–performance” relationship. To address this, we developed novel methods to conduct in-depth research on the microscopic properties of microspheres. Methods: Scanning electron microscopy (SEM) combined with a deep learning-based image segmentation (DLIS) algorithm was established for quantitative analysis of the pore structure. Laser confocal Raman spectroscopy (LCRS) was employed for in situ, non-destructive, three-dimensional (3D) visualization and quantitative mapping of the active pharmaceutical ingredient (API) distribution within microspheres. Results: This study successfully developed and applied SEM-DLIS and LCRS as reliable tools for microstructural and physicochemical characterization. SEM-DLIS analysis revealed significant differences in surface and internal pore structure among microspheres from different manufacturers and between particles of different sizes from the same batch. LCRS imaging further identified distinct API distribution patterns: uniform dispersion, outer-layer enrichment, and heterogeneous distribution. The combined data elucidate that the initial burst release is governed by the synergistic effect of surface porosity and API surface enrichment, whereas the sustained release kinetics are jointly regulated by the internal pore structure, particle size, and API spatial distribution. Conclusions: The findings establish that microstructure dictates release behavior and that all observed structural variations are linked to critical process parameters (CPPs), validating the “process determines structure” hypothesis. The established methodology provides a critical technical framework for the reverse engineering and quality equivalence assessment of generic microspheres, as well as for the quality-by-design-based optimization of innovative drug products, thereby advancing both pharmaceutical development and regulatory science.