Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 μs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.

The study evaluates cellulose nanocrystals (CNCs) as nanocarriers for targeted, intracellular delivery of molecular agents. CNCs were labeled with fluorescein-5′-isothiocyanate as an imaging agent and conjugated to folic acid (FA) as a targeting ligand. The CNC conjugates were characterized by UV–vis spectroscopy, ζ-potential analysis, dynamic light scattering, and atomic force microscopy. Cellular binding/uptake of the FA-conjugated CNCs by KB and MDA-MB-468 cells was quantified with cellular uptake assays. Internalization of the particles was confirmed by confocal microscopy. Uptake mechanisms were determined by inhibition studies with chlorpromazine and genistein. Binding affinity was qualitatively assessed with a free folate inhibition assay. Both KB and MDA-MB-468 cells exhibited significant and folate-receptor specific binding/uptake of FA-conjugated CNCs. Clathrin-mediated endocytosis was a significant uptake mechanism in both cell types, whereas caveolae-mediated endocytosis only played a significant role in MDA-MB-468 cells. Uptake inhibition of FA-conjugated CNCs by KB cells required high concentrations (>1 mM) of free FA. The observed FR-specific internalization of FA-conjugated CNCs by FR-positive cancer cells and tumors and their remarkable high affinity for the FR demonstrate the great potential of CNCs as novel nanocarriers for imaging agents and chemotherapeutics in the early detection and treatment of cancer.