Enhanced cytotoxicity of natural killer cells with Zn-alginate hydrogel microspheres

Natural killer (NK) cells are innate immune cells that exhibit nonspecific cytotoxicity against target cells and are known to have less immune rejection when allogeneic transplantation is performed. Therefore, various attempts are made to utilize them as a novel immunotherapeutic agent. However, there is a limitation that cytotoxicity is relatively low compared to T cells, so there is a need to sufficiently increase cytotoxicity to utilize NK cells as adaptive cell therapy. Zinc plays a crucial role in regulating signal transduction in immune cells by modulating the activity of key signaling molecules, such as NF-κB, acting as a second messenger. However, practical applications in cell culture step faced challenges due to (1) the cytotoxicity of zinc ions, (2) the formation of zinc oxide precipitates, and (3) the decrease in pH of the culture media. In this study, we hypothesized that effective delivery of zinc ions to NK cells would alter their signal transduction, thereby enhancing their target cell-killing activity. To test this, we synthesized zinc-alginate hydrogel microspheres (Zn-ALG) using alginate with divalent cations as crosslinkers and co-cultured these microspheres with NK cells to assess changes in killing activity. After co-culturing with Zn-ALG at a concentration of 1 bead/mL for approximately 72 h, we observed a significant decrease in the expression of inhibitory receptor genes TIM-3 and PDCD1 on NK cells by 21% and 28%, respectively. Additionally, the tumor cell-killing activity of NK cells increased by approximately 23%, showing statistically significant improvement.