Gemcitabine Delivery and Nucleolin-Targeting Nanocarriers Based on N6L-Functionalized Polyelectrolyte Complexes and Layer-By-Layer Lipid Nanoparticles

Abstract Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy due to dense stromal barriers. Although gemcitabine remains the standard treatment, its efficacy is limited. Combining it with the pseudopeptide N6L has shown synergistic effects, but administering them individually in biological systems poses significant challenges. To overcome this, we developed a polyanion−gemcitabine conjugate (PAAGem) coupled with N6L, a multivalent cationic ligand with intrinsic anticancer activity. The conjugation was confirmed, and gemcitabine retained its biological activity post-grafting. Quartz crystal microbalance studies revealed electrostatic interactions between PAAGem and N6L, guiding nanoparticle assembly. Building on this strategy, we engineered two electrostatic nanosystems: (i) a multilayer architecture constructed on nanostructured lipid carriers (NLCs) via a layer-by-layer approach, enabling controlled incorporation of cytotoxic agents, and (ii) a simpler polyelectrolyte complex (PEC) formed by spontaneous co-assembly in aqueous media. Colloidal analyses showed superior stability of LbL-NLCs. In murine PDAC cells, LbL-NLCs exhibited markedly enhanced anticancer efficacy compared with free gemcitabine + N6L, whereas PEC formulations provided limited improvement. These results were further validated in vivo. Overall, our study demonstrates that electrostatic multilayer architectures leveraging PAAGem−N6L interactions significantly outperform simple electrostatic complexes, highlighting LbL-functionalized NLCs as a promising nanotherapeutic strategy for PDAC treatment.