Preparation and purification of surfactant-free PLGA microspheres stabilized by bacterial cellulose nanocrystals for...

Surfactants are routinely used in polymeric microsphere fabrication but are difficult to remove and can compromise particle performance. Cellulose nanocrystals (CNCs) offer a sustainable alternative as Pickering stabilizers; however, their practical use in drug-loaded microspheres for biomedical applications has been hindered by the absence of an effective purification strategy. Here, we report a fully surfactant-free process for the preparation and purification of poly(lactic-co-glycolic acid) (PLGA) microspheres stabilized by bacterial cellulose nanocrystals (BCNCs). By exploiting the intrinsic density contrast between PLGA and BCNCs, a density-based centrifugation (DBC) method was developed to recover microspheres from excess BCNCs. An optimized 1.4 g/mL CaCl₂ separation medium achieved clear phase stratification and efficient microsphere recovery while preserving particle morphology. This purification strategy allowed systematic evaluation of BCNC concentration effects, revealing an optimal stabilization window that balanced emulsion stability, microsphere formation, and downstream recoverability. The platform was further validated using bovine serum albumin (BSA) as a model protein drug. BCNC-stabilized microspheres exhibited higher encapsulation efficiencies than conventional PVA-stabilized systems, consistent with the formation of a rigid Pickering barrier that reduced protein diffusion. Overall, this work introduces a CaCl₂-assisted DBC strategy that overcomes a key purification barrier in Pickering-emulsion-based microsphere fabrication, establishes BCNCs as practical particulate stabilizers for surfactant-free PLGA microsphere production with post-fabrication recoverability, and provides a broadly applicable purification platform for other particle-stabilized emulsion systems.