Background/Objectives: Electrochemotherapy (ECT) is a safe and efficient method of targeted drug delivery using pulsed electric fields (PEF), one that is based on the phenomenon of electroporation. However, the problems of electric field homogeneity within a tumor can cause a diminishing of the treatment efficacy, resulting only in partial response to the procedure. This work used gold nano-particles for electric field amplification, introducing the capability to improve available elec-trochemotherapy methods and solve problems associated with field non-homogeneity. Methods: We characterized the potential use of gold nanoparticles of 13 nm diameter (AuNPs: 13 nm) in combination with microsecond (0.6–1.5 kV/cm × 100 μs × 8 (1 Hz)) and nanosecond (6 kV/cm × 300–700 ns × 100 (1, 10, 100 kHz and 1 MHz)) electric field pulses. Finally, we tested the most prominent protocols (microsecond and nanosecond) in the context of bleomycin-based electrochemotherapy (4T1 mammary cancer cell line). Results: In the nano-pulse range, the synergistic effects (improved permeabilization and electrotransfer) were profound, with increased pulse burst frequency. Addi-tionally, AuNPs not only reduced the permeabilization thresholds but also affected pore resealing. It was shown that a saturated cytotoxic response with AuNPs can be triggered at significantly lower electric fields and that the AuNPs themselves are non-toxic for the cells either separately or in combination with bleomycin. Conclusions: The used electric fields are considered sub-threshold and/or not applicable for electrochemotherapy, however, when combined with AuNPs results in successful ECT, indicating the methodology’s prospective applicability as an anticancer treatment method.
