Optimization of Doxorubicin Loading into Citrus Lemon-Derived Nanoparticles and Evaluation of Their Antitumor Effects on Tumor Cell Lines.

Recently, lipid bilayer nanoparticles, similar to extracellular vesicles, have been identified in various plant juices and extracts. These plant-derived nanoparticles have attracted attention in the medical field for their intrinsic anti-inflammatory and antioxidant properties and for the advantages of inexpensive and abundant raw materials. Although citrus lemon-derived extracellular vesicle-like nanoparticles (cNPs) exert selective antitumor effects on several cancer cell lines, information on these effects remains limited. Therefore, in this study, we focused on cNPs as nanocarriers for effective drug delivery to cancer cells without introducing additional surface modification, with the aim of evaluating their intrinsic drug delivery capability. We optimized the conditions for loading doxorubicin hydrochloride (Dox), a model anticancer drug, into cNPs and successfully prepared Dox-loaded cNPs (Dox-cNPs) with high loading efficiency. In vitro cellular uptake of Dox-cNP-based samples by cancer cell lines was evaluated using confocal microscopy and flow cytometry, while their cytotoxicity was assessed using the Cell Counting Kit-8 assay. Phosphate-buffered saline-pretreated Dox-cNPs increased intracellular Dox fluorescence in rat glioblastoma (C6) cells. Notably, Dox-cNP-based samples exerted substantial cytotoxic effects not only on the rat C6 cell line but also on the human hepatoblastoma (HepG2) and breast cancer (MCF-7) cell lines. These findings suggest that cNPs may serve as potential nanocarriers for intracellular delivery of anticancer drugs.