Mechanoporation of Iron Oxide Nanoparticles into Supercharged Natural Killer Cells for MRI Detection

Poorly-differentiated hepatocellular carcinoma (HCC) is susceptible to natural killer (NK) cell therapy, making these patients good candidates for catheter directed NK cell immunotherapy. The FDA-approved iron-oxide nanoparticle, ferumoxytol, is typically used as a contrast agent for magnetic resonance imagining (MRI); however, it could also be used to label NK cells and confirm transcatheter delivery for improved treatment of HCC. This in vitro study tested the efficacy of mechanoporation for the delivery of ferumoxytol to primary supercharged NK cells (sNKs) for MRI detection.

 

Materials and Methods:

Supercharged NK cells were mechanoporated using the Gateway system from Portal Biotechnologies (Watertown, MA). The cells were mixed with the cargoes of interest, 3kDa fluorescent dextran (100 mg/mL) (ThermoFisher, Waltham, MA) and ferumoxytol (10, 100 μg/mL) (AMAG Pharmaceuticals, Inc., Waltham, MA), and pushed through a silicon chip containing pores of set sizes (6, 6.5, 7μm) at different pressures (10, 12 PSI). Viability and dextran delivery was measured using flow cytometry immediately post mechanoporation and 24 hours after. The Iron Stain Kit (Abcam, Waltham, MA) was used to confirm ferumoxytol delivery. All comparisons were performed with unpaired Student’s t-tests.



Results:

Supercharged NK cells had lower viability with smaller chips (6μm: 52.5±0.63%, p < 0.005; 6.5μm: 60.9±0.81%, p < 0.005; vs. 7μm: 67.8±0.83%, p < 0.005) but greater dextran delivery (6 mm: 55.2±7.3%, p < 0.005; 6.5μm: 41.4±8.5%, p < 0.005; 7μm: 37.8±5.8%, p < 0.05). Dextran delivery was higher with the 6μm chip at 12 PSI than at 10 (10 PSI: 53.6±8.9%, p < 0.05; 12 PSI: 56.7±8.3%, p < 0.05). The Iron Staining Kit confirmed iron delivery in sNKs mechanoporated at 12 PSI with a 6 μm chip and 10 μg/mL ferumoxytol.

Conclusion: Given ferumoxoytol’s FDA approval, iron-labeled sNKs have the potential to be given as an immunotherapy treatment for poorly-differentiated HCC and be visualized on MRI to confirm direct catheter delivery. This advancement can improve tumor targeting and requires less resources than systemic delivery of NK cell immunotherapy. In this study, dextran delivery was observed in the mechanoporated sNKs. Viability and delivery is tunable by adjusting pressure and pore size. Maximum delivery of dextran was observed using the 6 μm pore size chip at 12 PSI. Few cells were stained with the Iron staining kit post mechanoporation. To improve ferumoxytol delivery using this method, optimization using a larger cargo molecule, such as fluorescent antibody, may better inform the optimal conditions for ferumoxytol.