Enhanced Therapeutic Efficacy Against Melanoma through Exosomal Delivery of Hesperidin

Melanoma, characterized as the most aggressive and metastatic form of skin cancer, currently has limited treatment options, predominantly chemotherapy and radiation therapy. However, the drawbacks associated with parenterally administered chemotherapy underscore the urgent need for alternative compounds to combat melanoma effectively. Hesperidin (HES), a flavonoid present in various citrus fruits, exhibits promising anticancer activity. Nevertheless, the clinical utility of HES is hindered by challenges such as poor water solubility, a short half-life, and low oral bioavailability. In response to these limitations, we introduced a novel approach by formulating HES-loaded exosomes (Exo-HES). Isolation of exosomes was achieved through the ultracentrifugation method, and HES was efficiently loaded using the sonication method. The resulting formulations displayed a desirable particle size (∼106 nm) and exhibited a spherical morphology, as confirmed by scanning electron and atomic force microscopy. In vitro studies conducted on B16F10 cell lines demonstrated higher cytotoxicity of Exo-HES compared to free HES, supported by enhanced cellular uptake validated through coumarin-6-loaded exosomes. This superior cytotoxicity was further evidenced by DNA fragmentation, increased generation of free radicals (ROS), loss of mitochondrial membrane potential, and effective inhibition of colony formation. The antimetastatic properties of Exo-HES were confirmed through wound healing and transwell migration assays. Oral pharmacokinetics studies revealed a remarkable increase of approximately 2.5 times in oral bioavailability and half-life of HES when loaded into exosomes. Subsequent in vivo experiments utilizing a B16F10-induced melanoma model in Swiss mice established that Exo-HES exhibited superior anticancer activity compared to HES after oral administration. Importantly, no biochemical, hematological, or histological toxicities were observed in tumor-bearing mice treated with Exo-HES. These findings suggest that exosomes loaded with HES represent a promising nanocarrier strategy to enhance the therapeutic effectiveness of hesperidin in melanoma treatment.