Associate Scientist University of Florida Niceville, Florida, United States
Introduction:: The development of cancer immunotherapies in recent years has seen an exciting degree of growth and promising results in clinical trials. However, the success of T cell focused strategies has been limited by safety concerns and the high cost of cell sourcing and expansion. Natural killer (NK) cells have potential to be an “off-the-shelf” alternative to T cell-based therapies given their innate ability to target and destroy many varieties of cancerous tissue without the need for prior sensitization. NK cells also serve a crucial role in activating the adaptive immune system against tumors, making them an ideal target for strategies seeking to improve efficiency of existing T-cell strategies. Recently there has been a growing interest in using nanoparticles to manipulate and improve NK cell function and tracking. Our group has explored the application of manganese dioxide nanoparticles (MnO2) for cytoprotection of NK cells and RNA delivery. Despite growing interest in the field, little is understood about how NK cells take up nanoparticles. While many are employing nanoparticles to manipulate NK cells, the explicit mechanisms of endocytosis that various nanoparticle delivery systems undergo in NK cells are left unexplored. The objective of this project was to determine the primary uptake pathway utilized by MnO2 NPs while providing novel documentation for future uptake studies.
Materials and Methods:: MnO2 NPs were synthesized according to previously reported methods. Briefly, potassium permanganate and poly allylamine hydrochloride were mixed in a redox reaction producing MnO2 NPs. These particles were conjugated with PEG (MW 3400) to improve colloidal stability and then fluorescently tagged with Alexa 488. The NK-92 cell line was used in this study given its success in clinical trials as an allogenic source of NK cells. MnO2 NP uptake was visualized via confocal microscopy and quantified via flow cytometry. The energy dependence of MnO2 NP uptake was measured by measuring uptake of nanoparticles at 4C and 37C. Finally, uptake pathways were selectively inhibited with small molecule inhibitors (10ug/mL methyl-B-cyclodextrin, 40ug/mL amiloride hydrochloride, and 10ug/mL chlorpromazine hydrochloride) to observe subsequent changes in uptake profile. NK cells were co-cultured with MnO2 NPs and individual endocytosis inhibitors up to 6 hours in low serum media. Cells were fixed in 10% buffered formalin phosphate and stained with a fixable Live/Dead violet as a control to ensure cell viability across all experimental conditions.
Results, Conclusions, and Discussions:: Although not widely studied, it was evident from this study that MnO2 NPs could be readily taken up by NK cells in standard culture conditions. 90% of cells were positive for MnO2 presence following 1 hour of incubation. Further, there was no loss of cell viability with the addition of the MnO2 NPs. However, incubation at 4C showed a 94% reduction in MnO2 uptake into NK cells, indicating the more prominent role of active transport mechanisms in MnO2 NP uptake. Following selective endocytosis inhibition at 37C, our findings show that clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis inhibition all decreased MnO2 NP uptake after 1 hour of co-incubation. However, after 3 and 6 hours, only clathrin-mediated endocytosis inhibition continued to show decreased MnO2 NP uptake percentage. This may indicate that other types of inhibition can be overcome with time, but through what mechanism this occurs is not clear. But, clathrin-mediated endocytosis may have a more significant role as the primary pathway across the cell membrane for MnO2 NPs. With this in mind, we hope to explore the eventual fate of these nanoparticles in the cell following internalization. Trafficking of these particles and their potential cargo will be critical information to accurately deliver small molecules or genetic material for the eventual manipulation of NK cells. These methods and results serve as a starting point to inform the design of nanoparticle systems for NK cell manipulation.
