Development of an active loading strategy for methylene blue in liposomes and its application in EGFR-targeted photodynamic therapy.

As an ancient drug, methylene blue (MB) has shown promise in tumor photodynamic therapy (PDT) due to its excellent light-induced reactive oxygen species (ROS) yield. However, its rapid plasma clearance and lack of tumor targeting have limited its application in this field. To overcome these limitations, we developed MB-loaded liposomes using an active loading method via an ammonium sulfate gradient, achieving a high encapsulation efficiency of 83.57%, which is significantly higher than that of conventional passive loading (37.50%). The optimized liposomal formulation (Lip@MB) exhibited a uniform particle size (∼120 nm), improved colloidal stability, and reduced drug leakage under both physiological and storage conditions compared to passively loaded liposomes (Lip@MB-Passive). Pharmacokinetic studies in mice confirmed that Lip@MB significantly prolonged the plasma circulation time of MB. Furthermore, surface modification with an epidermal growth factor receptor (EGFR)-targeting affibody (ZEGFR) yielded a targeted formulation (Lip@MB-ZEGFR), which promoted enhanced cellular uptake in EGFR-overexpressing A431 cells. This targeting effect resulted in more efficient light-induced ROS generation and superior photodynamic cytotoxicity, with an IC50 of 0.38 µM, substantially lower than that of the non-targeted Lip@MB (IC50 = 2.04 µM). In summary, the combination of active loading and tumor-targeting modification offers a promising strategy to enhance the therapeutic performance of MB for targeted PDT applications.