2.1. Concept of EMP

EMP induces membrane perforation by combining electrical and mechanical stimuli. EMP generates pressure fluctuations through changes in the microbubble volume. This pressure fluctuation transiently depolymerizes the cytoskeleton that supports the cell membrane. As a result, the cell membrane of EMP-treated cells becomes more capable of maintaining pores compared to untreated cells, thereby facilitating material delivery into the cells (Figs. 3(a) and (b)) ¹³.

When EMP is applied to cells, (1) pores are formed on the cell membrane; (2) external materials are taken up into the cytoplasm; and (3) the cell recovers its membrane, which in turn closes the pores and retains the materials inside the cell. This method can be used not only for genetic materials such as plasmids, but also for artificial microparticles (Figs. 3(c)–(e)). By introducing artificial materials into cells using this approach, it is possible to evaluate their compatibility at the cellular level.

2.2. Instrumentation for EMP

EMP was performed using a bubble injector, as shown in Fig. 4. The injector in this study had a core–shell structure, consisting of a tungsten electrode of 400-μm diameter and a glass capillary to insulate the electrode. When the injector is immersed in an aqueous solution, such as a cell suspension, and a high-voltage pulse is applied, a strong electric field is generated near the tip of the electrode. Simultaneously, the intense electric field at the electrode tip induces the formation of a microbubble. This microbubble changes its volume in response to the applied electric pulses and intervals (Fig. 3(b)). This oscillation of microbubble volume generates pressure fluctuations in the liquid, thereby altering the state of the cytoskeleton ¹⁴.

Figure 5 shows the entire arrangement of the bubble injection system. The cathode of the power supply was connected to the bubble injector and the anode was connected to a stainless-steel counter electrode attached to the outer shell of the bubble injector. The voltage and current applied to the bubble injector were monitored by using an oscilloscope. A high-voltage power supply (Hyfrecator 2000, CONMED, Largo, FL, USA) was used as the pulse source to control the electrical power during EMP. The number of electrical pulses was controlled using a microcontroller (Uno-R3, Arduino, Monza, Italy).

2.3. Material Delivery to Adherent Cells

To investigate the delivery of artificial materials by EMP, HeLa cells, a type of adherent cells, were used as the target of material delivery. A total of \(1.5 \times 10^6\) HeLa cells were suspended in 4.5 μL of culture medium (OptiMEM, Thermo Fisher Scientific, Waltham, MA, USA) to prepare a cell suspension. Immediately before EMP treatment, the suspension was mixed by tapping to ensure that both electrical and mechanical stimuli reached as many cells as possible.

During EMP treatment, the pulse generator was operated in bipolar mode, and an electrical power of 12 W was applied. Each pulse cycle consisted of a 1.1-μs pulse followed by a 30-μs interval. One set comprised 1,000 pulse cycles, and 20 sets were applied in a single EMP treatment, corresponding to 20,000 electrical pulses delivered to the cell suspension. A 2-s pause was introduced between sets. These EMP parameters were selected based on our previous studies ^13,14, in which energy input of 1.1-μs pulse width with 30-μs intervals generated microbubbles on the order of 100 μm and effectively induced membrane permeabilization in adherent cells without markedly reducing cell viability.

During EMP treatment, the voltage and current waveforms were continuously monitored using an oscilloscope. Representative voltage and current waveforms obtained during the normal EMP operation are shown in Fig. 6.

Fluorescent polystyrene microbeads (2, 3, 6, and 10 μm in diameter; Fluoresbrite, Polysciences, Warrington, PA, USA) and NPCs ⁷ were used as artificial materials for EMP-mediated delivery. After EMP treatment, the treated cell suspension was diluted into a culture medium, OptiMEM, containing 10% (v/v) fetal bovine serum (Biosera, Cholet, France), at a ratio of \(1:99\) (v/v) and incubated at 37°C in a 5% CO\(_2\) incubator.

The material-loaded cells were stained for evaluation using fluorescence microscopy. Propidium iodide (PI) (Dojindo, Kumamoto, Japan) was used to stain dead cells. Hoechst 33342 (Dojindo) was used to stain cell nuclei, and 5-chloromethylfluorescein diacetate (CMFDA) (Thermo Fisher Scientific, Waltham, MA, USA) was used for cytoplasmic staining.

3.1. Bubble Formation During EMP

The generation of microbubbles responsible for the mechanical stimulus during EMP is shown at the macroscopic level in Fig. 7. Upon applying electrical pulses during EMP, microbubble formation was accompanied by light emission. The generated bubbles were ejected downward from the electrode surface to the liquid phase. Subsequently, the bubbles rose toward the liquid surface owing to buoyancy. Microscopic images of bubble formation were obtained using a high-speed camera (HPV-X2, Shimadzu, Kyoto, Japan) (Fig. 8). The application of multiple electrical pulses generates small microbubbles on the electrode surface. The continuous application of pulses increased the bubble volume, leading to the formation of an integrated microbubble. This microbubble subsequently collapsed and was ejected from the electrode surface while generating a microjet. This cycle of microbubble formation and ejection occurred multiple times within one set of applied pulses (1,000 pulses).

3.2. Results of Bead Injection

Fluorescent polystyrene beads were delivered to HeLa cells using EMP. After bead delivery, cells were cultured for 24 h. Before observation under a fluorescence microscope, the culture medium containing free polystyrene beads was aspirated, and the cells were washed with PBS to remove beads that were not taken up by the cells. Washed HeLa cells were observed using fluorescence microscopy. Cell nuclei were stained with Hoechst 33342 to identify HeLa cells using blue fluorescence. Bright-field images were used to determine cell boundaries, and cells containing green fluorescent polystyrene beads within the cell contour were counted. To confirm that the beads were internalized, rather than attached to the cell surface, confocal microscopy was used to obtain cross-sectional images along the \(z\)-axis (Fig. 9).

The correlation between bead diameter and delivery efficiency is shown in Fig. 10. Delivery efficiency was calculated as the number of bead-containing cells divided by the total number of cells. Bead-containing cells were defined as those in which both polystyrene beads with green fluorescence and nuclei with blue fluorescence were observed within the cell boundary. The total number of cells was determined based on the number of nuclei.

Beads with diameters up to 3 μm exhibited high delivery efficiency into HeLa cells, whereas beads with diameters exceeding 6 μm showed a significant decrease in delivery efficiency. The diameter of non-adherent HeLa cells during EMP treatment was estimated to be 17 μm ^16,17. Assuming HeLa cells were spherical, a membrane opening sufficient for a 3-μm particle would correspond to 0.78% of the cell surface area, whereas that for a 6-μm particle would correspond to 3.1% of the cell surface area. These results suggest that HeLa cells readily take up objects approximately up to 0.8% of their surface area. The selected EMP conditions may have supported the uptake of micrometer-scale particles through the combined effects of electrical and mechanical stimulation. Electrical stimulation can increase membrane permeability, whereas mechanical stimulation generated by oscillatory changes in microbubble volume may transiently perturb the cytoskeleton and facilitate membrane deformation. Such transient membrane deformation would be beneficial for the entry of particles to approximately 3 μm in diameter. In contrast, particles exceeding 6 μm would require larger membrane openings and greater membrane deformation, which may explain the marked decrease in delivery efficiency.

Cell viability was also evaluated 24 h after bead delivery (Figs. 10(a) and (b)). Viability was calculated based on the number of dead cells relative to the total number of cells; dead cells were defined as red fluorescent (PI\(^{+}\)) cells; 24-h cell viability was comparable regardless of bead size. This suggests that the polystyrene microparticles did not exhibit cytotoxicity toward HeLa cells within the range of tested bead sizes.

3.3. Results of Silver-Coated DNA-Functionalized Gold Nanoparticle Crystals (Au@Ag NPC)

Au@Ag NPCs were delivered into HeLa cells using EMP under the same conditions as those used for polystyrene bead delivery. Based on the bead delivery results, NPC diameter was adjusted to approximately 1–1.5 μm to facilitate uptake. Aggregated Au@Ag NPCs exceeding 6 μm in diameter were excluded from counting, as they were expected to exhibit low delivery efficiency.

After NPC delivery, the cells were cultured for 1–4 d. Prior to fluorescence observation, the culture medium containing free Au@Ag NPCs was aspirated, and the cells were washed with PBS to remove Au@Ag NPCs that were not taken up by the cells. The washed cells were observed using fluorescence microscopy (Fig. 11). The cytoplasm was stained with CMFDA to show green fluorescence, and cells containing non-fluorescent Au@Ag NPCs within the cytoplasm were counted. Bright-field images of the same samples were used to confirm the presence of NPCs in the cells.

After 24 h of culture, NPC-containing cells accounted for 0.50% of the total cell population. Of these, 61% was attributed to EMP-mediated delivery, whereas the remaining 39% was considered to result from spontaneous cellular uptake, which occurred to cells with no EMP treatment.

The lower delivery efficiency of Au@Ag NPCs compared to that of polystyrene beads may be attributed to differences in mechanical stiffness. Polystyrene beads have a Young’s modulus in the GPa range ¹⁸, whereas Au@Ag NPCs are crosslinked by DNA strands between nanoparticles and are therefore expected to have lower mechanical stiffness ^19,20. During microbubble collapse, rigid polystyrene beads may transfer momentum more effectively to the cell membrane, whereas softer Au@Ag NPCs may undergo deformation. Such a deformation could dissipate a fraction of the impact energy and reduce the force transmitted to the membrane, resulting in a lower delivery efficiency. The optimization of the EMP parameters for Au@Ag NPC delivery requires further investigation. Nevertheless, because NPC delivery via EMP was confirmed despite the difference in stiffness, the cytotoxicity of Au@Ag NPCs was evaluated.

The number of dead cells after Au@Ag NPC delivery is shown in Fig. 12. Because Au@Ag NPCs remained in the culture environment after treatment, this experiment enabled evaluation of potential acute toxicity of Au@Ag NPCs. HeLa cells were divided into four groups based on the presence or absence of Au@Ag NPC and EMP. No significant differences were observed in the number of dead cells among the four groups. In addition, no substantial change in cell death was observed over 1–4 d in any group. These results indicate that the presence of Au@Ag NPCs in the culture environment did not induce acute cytotoxicity in HeLa cells. It has also been demonstrated that EMP treatment stimulate cells within a recoverable range. Thus, EMP-mediated Au@Ag NPC delivery, which combines a low-cytotoxicity material with a mild delivery method, resulted in minimal acute cytotoxicity.