Comparison of the Cytotoxicity, Internalization and Anti-Cancer Drug Delivery Efficacy of Nature Killer Cell Derived Nanovesicles and Extracellular Vesicles

Purpose: Natural killer (NK) cell-derived extracellular vesicles (NK-EVs) have garnered significant research interest in the field of tumor immunotherapy. However, the large-scale production of NK-EVs remains a major challenge, limiting their clinical application. This study aims to develop a simple and efficient method for the preparation of NK cell-derived nanovesicles (NK-NVs) and to evaluate their cytotoxicity and drug delivery potential. Methods: In this study, we efficiently produced large quantities of NK-NVs by extruding NK cells. We conducted comprehensive characterization and protein profiling analyses of NK cells, NK-EVs, and NK-NVs. The cytotoxicity and cellular uptake of NK-NVs were evaluated, and the internalization mechanism was explored. To assess the drug delivery capability, doxorubicin (DOX) was loaded into NK-NVs (NK-NVs-DOX) using various loading strategies, including co-incubation, sonication, extrusion, and electroporation. We thoroughly evaluated the drug loading efficiency, particle size, stability, and cytotoxicity of NK-NVs-DOX. Results: Extrusion-derived NK-NVs exhibited a remarkable 402.18-fold increase in particle yield and a 325.76-fold enhancement in protein yield compared to ultracentrifugation-isolated NK-EVs, while maintaining comparable morphology and EV-specific markers (Alix, TSG101, CD9). Functionally, NK-NVs induced delayed cytotoxicity against cancer cells via caveolin-mediated endocytosis, selectively sparing normal cells. Proteomic analysis revealed that NK-NVs shared 7,366 proteins with NK cells, surpassing the 5,326 proteins found in NK-EVs. Furthermore, extrusion-optimized NK-NVs-DOX demonstrated pH-sensitive drug release (30% higher at pH 5.5), significantly enhanced anti-cancer effects across four cancer cell lines, and stable drug retention for up to 28 days at 4°C, highlighting their promising therapeutic potential. Conclusion: Extrusion-derived NK-NVs offer a low-cost, rapid, and high-yield production method while selectively inducing cytotoxicity in cancer cells. Their pH-sensitive drug release enhances drug loading stability. These advantages establish NK-NVs as a promising and scalable platform for tumor immunotherapy and drug delivery with significant clinical potential. Keywords: natural killer cells, nanovesicles, extracellular vesicles, internalization, drug delivery, antitumor therapy