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As an increasing number of studies use gold nanoparticles (AuNPs) for potential medicinal, biosensing and therapeutic applications, the synthesis and use of readily functional, bio-compatible nanoparticles is receiving much interest. For these efforts, the particles are often taken up by the cells to allow for optimum sensing or therapeutic measures. This process typically requires incubation of the particles with the cells for an extended period. In an attempt to shorten and control this incubation, we investigated whether nanosecond pulsed electric field (nsPEF) exposure of cells will cause a controlled uptake of the particles. NsPEF are known to induce the formation of nanopores in the plasma membrane, so we hypothesized that by controlling the number, amplitude or duration of the nsPEF exposure, we could control the size of the nanopores, and thus control the particle uptake. Chinese hamster ovary (CHO-K1) cells were incubated sub-10 nm AuNPs with and without exposure to 600-ns electrical pulses. Contrary to our hypothesis, the nsPEF exposure was found to actually decrease the particle uptake in the exposed cells. This result suggests that the nsPEF exposure may be affecting the endocytotic pathway and processes due to membrane disruption.

The underlying mechanism(s) responsible for nanoporation of phospholipid membranes by nanosecond pulsed electric fields (nsEP) remains unknown. The passage of a high electric field through a conductive medium creates two primary contributing factors that may induce poration: the electric field interaction at the membrane and the shockwave produced from electrostriction of a polar submersion medium exposed to an electric field. Previous work has focused on the electric field interaction at the cell membrane, through such models as the transport lattice method. Our objective is to model the shock wave cell membrane interaction induced from the density perturbation formed at the rising edge of a high voltage pulse in a polar liquid resulting in a shock wave propagating away from the electrode toward the cell membrane. Utilizing previous data from cell membrane mechanical parameters, and nsEP generated shockwave parameters, an acoustic shock wave model based on the Helmholtz equation for sound pressure was developed and coupled to a cell membrane model with finite-element modeling in COMSOL. The acoustic structure interaction model was developed to illustrate the harmonic membrane displacements and stresses resulting from shockwave and membrane interaction based on Hooke’s law. Poration is predicted by utilizing membrane mechanical breakdown parameters including cortical stress limits and hydrostatic pressure gradients.

Second Harmonic Generation (SHG) imaging is a useful tool for examining the structure of interfaces between bulk materials. Recently, this technique was applied to detecting subtle perturbations in the structure of cellular membranes following nanosecond pulsed electric field (nsPEF) exposure. Monitoring the cell’s outer membrane as it is exposed to nsPEF via SHG has demonstrated that nanoporation is likely the root cause for size-specific, increased cytoplasmic membrane permeabilization. It is theorized that the area of the membrane covered by these pores is tied to pulse intensity or duration. The extent of this effect along the cell’s surface, however, has never been measured due to its temporal brevity and minute pore size. By enhancing the SHG technique developed and elucidated previously, we are able to obtain this information. Further, we vary the pulse width and amplitude of the applied stimulus to explore the mechanical changes of the membrane at various sites around the cell. By using this unique SHG imaging technique to directly visualize the change in order of phospholipids within the membrane, we are able to better understand the complex response of living cells to electric pulses.

Nanosecond pulsed electric fields (nsPEF) cause the formation of small pores, termed nanopores, in the membrane of cells. Current nanoporation models treat nsPEF exposure as a purely electromagnetic phenomenon, but recent publications showing pressure transients, ROS production, temperature gradients, and pH waves suggest the stimulus may be physically and chemically multifactorial causing elicitation of diverse biological conditions and stressors. Our research group's goal is to quantify the breadth and participation of these stressors generated during nsPEF exposure and determine their relative importance to the observed cellular response. In this paper, we used advanced imaging techniques to identify a possible source of nsPEF-induced acoustic shock waves. nsPEFs were delivered in an aqueous media via a pair of 125 μm tungsten electrodes separated by 100 μm, mirroring our previously published cellular exposure experiments. To visualize any pressure transients emanating from the electrodes or surrounding medium, we used the Schlieren imaging technique. Resulting images and measurements confirmed that mechanical pressure waves and electrode-based stresses are formed during nsPEF, resulting in a clearer understanding of the whole exposure dosimetry. This information will be used to better quantify the impact of nsPEF-induced acoustic shock waves on cells, and has provided further evidence of non-electrical-field induced exposures for elicitation of bioieffects.