PEGylated liposomes co-encapsulating darolutamide and hesperetin for enhanced prostate cancer therapy: 2D, 3D PC3 models and in-vivo evaluation.

In this study, a darolutamide and hesperetin co-loaded PEGylated liposomal formulation (DHLP) was developed for the treatment of non-metastatic castration-resistant prostate cancer (nmCRPC). The formulation was prepared using the ethanol injection method, followed by membrane extrusion through a 100 nm polycarbonate filter to ensure size uniformity. The optimized DHLP liposomal formulation exhibited a clear bluish appearance, with a mean particle size of 106.5 ± 3.2 nm, a polydispersity index of 0.128 ± 0.023, and a zeta potential of -14.10 ± 0.25 mV, indicating colloidal stability and uniformity. Spherical morphology was confirmed through microscopic analysis. High encapsulation efficiencies were achieved for both DA (90.1 ± 3.3 %) and HE (91.3 ± 2.7 %). For both medication, in vitro drug release experiments showed a sustained release profile from the DHLP. Cytotoxicity assessment in PC3 prostate cancer cells revealed a 2.83-fold increase in cytotoxicity compared to free DA solution, a 3.89-fold increase compared to free HE solution, and a 1.47-fold improvement over the combined DA-HE solution. The combination index value of 0.89 confirmed synergistic anticancer activity. DHLPs inhibited cell migration, increased ROS generation, and reduced 3D spheroid size, with enhanced cellular uptake contributing to improved therapeutic efficacy. In-vivo pharmacokinetic studies in BALB/c mice demonstrated a 1.7- and 3.4-fold increase in the plasma half-life of DA and HE, respectively, compared to their free drug solutions. Biodistribution studies revealed reduced accumulation of DHLP in vital organs compared to the free drug solution. Acute toxicity assessment at three dose levels showed no histopathological alterations, indicating a favorable safety profile. These results imply that DHLP is a potential nanocarrier system for the effective management of prostate cancer.