Collagenase-Functionalized Liposomes Overcome Stromal Barriers in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) features a dense desmoplastic stroma dominated by type I collagen, which forms a physical barrier that limits vascular perfusion and drug penetration, resulting in poor therapeutic outcomes. Here, we report gemcitabine-loaded collagenase-conjugated liposomes (GLCLs) that enzymatically remodel the collagen-rich extracellular matrix (ECM) while delivering a cytotoxic payload. GLCL retained enzymatic activity and stability, exhibited prolonged circulation, and achieved superior tumor accumulation with deeper and more homogeneous intratumoral gemcitabine penetration than nonfunctionalized liposomes. In PDAC-bearing mice, GLCL showed a 6-fold higher tumor growth inhibition (69.8%) than gemcitabine-loaded liposomes without collagenase (GLL; 10.9%) at the same dose. Importantly, this work provides the first molecular-level validation of nanocarrier-mediated drug penetration in PDAC, achieved through a multiscale approach combining <i>in vivo</i> imaging, <i>ex vivo</i> fluorescence, and quantitative desorption electrospray ionization mass spectrometry imaging (DESI-MSI). GLCL enzymatically overcomes PDAC's fibrotic collagen barrier, offering a clinically translatable strategy for deep intratumoral drug delivery.