Liposomal particokinetics and intratumoral microdistribution: A quantitative reassessment of the enhanced permeability and retention effect.

The enhanced permeability and retention (EPR) effect, a cornerstone of cancer nanomedicine, has been predominantly interpreted at the macroscopic level, offering limited insight into the microscopic dynamics governing nanocarrier delivery. To address this gap, we directly compared the intratumoral particokinetics of liposomal carriers with that of their drug payload (doxorubicin) through precise tracing and quantitative analysis. Using a second near-infrared (NIR-II) aggregation-caused quenching (ACQ) probe, ACQ5, we achieved accurate in vivo tracking of doxorubicin-loaded liposomes. This approach minimizes artifacts from free labels and ensures superior fluorescence linearity. Our results show substantial sequestration of liposomes by the mononuclear phagocyte system (liver: 16.3% ID/g at 8 h; spleen: 70.5% ID/g at 36 h), with only modest tumor accumulation (∼4% ID/g after 24 h). Importantly, by noninvasively differentiating intravascular from extravascular signals, we established that microdistribution serves as a more meaningful indicator of permeation efficiency than total tumor accumulation. Moreover, we revealed a critical spatial dissociation: although liposomes progressively penetrated the tumor interstitium, doxorubicin readily dissociated and was largely retained in perivascular regions. This study challenges the classical macro-level EPR concept and underscores the necessity of a microscopic reassessment of intratumoral particokinetics to guide the development of next-generation nanocarriers with improved therapeutic efficacy.