Abstract
The clinical success of PEGylated liposomal doxorubicin (DOX) is fundamentally attributed to its unique internal nanocrystalline structure. However, a notable discrepancy exists between the rapid drug uptake predicted by loading kinetics and the extended incubation times employed in actual manufacturing. By integrating in situ time-resolved synchrotron small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and in vitro release kinetics analysis, this study systematically elucidates the multi-step structural evolution and formation mechanism of DOX-sulfate nanocrystals during remote loading via the ammonium sulfate gradient method, along with its correlation with the final functional performance of the formulation. Our results demonstrate that the rapid drug uptake and achievement of target encapsulation efficiency (EE%) do not necessarily indicate the formation of a mature, stable nanocrystalline structure. Instead, during a critical post-loading maturation stage, the intraliposomal nanocrystals undergo gradual lattice optimization into a thermodynamically stable structure. Liposomes containing "sub-mature" aggregates exhibit pronounced burst leakage, whereas those with fully matured nanocrystals ensure near-zero-order release kinetics and superior stability. These observations provide useful insights that may inform future process optimization and offer a potential basis for structural quality control of liposomal nanodrugs.