Affordable handheld devices using multiple electric pulses for easy delivery of genetic material into skin

Voltage and current profiles from different pulsers were recorded using a digital oscilloscope (SDS 1202X‐E, Siglent, Shenzhen, China) at a sampling rate of 25 MHz. Voltage measurements made across an open circuit were obtained using a 1000X high‐voltage probe connected between the oscilloscope and the pulser (Supplementary Figure ). Voltage and current measurements were made across skin by pressing a MEA into porcine skin ex vivo (Pel‐Freeze, Rogers, AR), measuring voltage across the skin, and determining current by measuring the voltage across a 100‐Ω resistor placed in series with the skin; the current was calculated as the voltage drop across the resistor divided by resistance. S1

Transient force profiles during pulser actuations were measured using a single‐point load cell (20 kg, RSP1‐020M‐A CO1, Loadstar Sensors, Fremont, CA), with data collected at a 10 kHz sampling rate using the Loadstar software.

All animal experiments were conducted in accordance with ethical approval from the Institutional Animal Care and Use Committee (IACUC, protocol # A100427) of the Georgia Institute of Technology. BALB/c mice (female, 7–18 weeks old) and adult Wistar rats (female, 6–8 weeks old) were obtained from Charles River Laboratories (Charles River, Wilmington, MA) and housed in a pathogen‐free facility at the Georgia Institute of Technology under a 12 h light/12 h dark schedule (lights on at 07:00, off at 19:00), with water and chow available ad libitum. For this feasibility study, we did not perform power calculations to determine sample size or use an animal randomization schedule. Investigators were not blind to the experimental groups. The ARRIVE guideline summary for the animal study is provided in the supplementary information.

Mice and rats were prepared under anesthesia 1 day prior to ID injections by removing hair on their dorsal skin with clippers, followed by a 1–2‐min application of depilatory cream (Nair, Church & Dwight, Ewing, NJ), which was then cleaned off with lukewarm water. Animals were anesthetized in an induction charged with 5% isoflurane in O₂ by isoflurane vaporizer (SurgiVet Model 100, Smiths Medical, Dublin, OH), then fitted with a standard rodent mask and maintained under general anesthesia with 1%–2% isoflurane during the procedures.

Twenty microliters of phosphate‐buffered saline (PBS) free of Ca²⁺ and Mg²⁺ and containing firefly‐luciferase encoding N1‐methylpseudouridine (m¹Ψ)‐modified naked mRNA (0.25 μg/μL, Genscript, Piscataway, NJ) with RNase inhibitor (1 U/μL, RNasin Plus; Promega, WI), or gWiz‐GFP plasmid (2.5 μg/μL, Aldevron, Freiburg, Germany) was intradermally injected using a 31G insulin syringe (Easytouch U‐100, MHC medical products, Fairfield, OH), forming a visible bleb at the skin surface of mice and rats, respectively (Supplementary Figure S2). Immediately after the injection, 10 electric pulses were administered through MEA insertion at the bleb site. For ePatch and the eIgniter, pulses were triggered manually by clicking the buttons on the pulsers, with ~1 s between pulses, respectively. For RotoPatch, an Arduino‐controlled script automated pulse delivery with a 300 ms interval between successive pulses (Supplementary Figure S3).

Bioluminescence and fluorescence imaging were used, respectively, to quantify the expression kinetics of firefly luciferase mRNA and green fluorescent protein (GFP) plasmid in mice and rats. Prior to bioluminescence imaging, mice were injected intraperitoneally with D‐luciferin (200 μL, 15 mg/mL; Gold Biotechnology, Olivette, MO) and were imaged within 15–25 min using the IVIS Lumina II system (PerkinElmer, Waltham, MA). Total photon flux (bioluminescence, in mice) and radiant efficiency (fluorescence, in rats) were quantified using Living Image Software (v4.3.1, Caliper Life Sciences, Hopkinton, MA) within a fixed circular region of interest at each ID injection site. Imaging was conducted on multiple days post‐injection to measure the expression kinetics of naked mRNA and GFP‐plasmid with and without electroporation, with ID injected PBS as a negative control. To quantify protein and gene expression from mRNA and plasmid DNA, we calculated the area under the expression curve using the trapezoidal rule in MATLAB (Supplementary Figure ). S4

Statistical analysis was performed in Prism 5.3 software (GraphPad, San Diego, CA). The Shapiro–Wilk test was performed to check the normality of the in vivo protein expression data for different study groups. For mRNA data, one group did not meet the assumption of normality; therefore, the Friedman test followed by Dunn's post‐hoc multiple comparisons was used for statistical comparison. In contrast, for normally distributed plasmid DNA data, repeated measures analysis with Dunnett's multiple comparisons test was used. Statistical significance was established with p‐values less than 0.05.

We previously developed ePatch as a means to generate electroporation pulses using a small, low‐cost, hand‐held device that leverages the piezoelectric element of a barbeque lighter to produce electric pulses30 (Figure 1a). While effective, the ePatch is only able to administer one electric pulse for each manual actuation of the device. Because electroporation often benefits from giving multiple pulses, a user needs to repeatedly actuate ePatch each time a pulse is given. The force of actuation to energize the piezoelectric element to produce an electrical discharge when using ePatch is also relatively high, leading to possible user fatigue.

To address these limitations, we developed RotoPatch (Figure 1b–d), which consists of 3D‐printed components and a casing that houses a spring, a piezoelectric crystal, and a MEA (Figure 1f). Its rotational actuation mechanism enables pulse generation with a low tangential force, nearly 10 times lower than the normal force required to actuate a single click of the ePatch to generate a single pulse (Supplementary Figure S5), rendering the RotoPatch more user‐friendly for clinical operators.

The pulse generation of the RotoPatch involves conversion of rotational energy into repeated translational strikes through a simple mechanical sequence (Supplementary Figure ). As the cap and rotor rotate, interfacing angled ridges on both the rotor and the hammer assembly cause the hammer assembly to lift vertically, compressing the spring until it reaches the ridge peak. Immediately after passing the peak, the hammer is suddenly released and strikes a metal pin positioned above the piezoelectric crystal, generating an electric pulse. This sequence repeats multiple times per rotation, producing a consistent train of pulses. S6

We developed a family of three distinct versions of the RotoPatch: (i) a manual RotoPatch, operated by hand rotation similar to loosening the lid on a jar (Figure 1b); (ii) a manual RotoPatch with a handle, operated by rotation of a foldable handle similar to a manual coffee grinder handle (Figure 1c); and (iii) a motorized RotoPatch, powered by a stepper motor controlled by a microcontroller that is activated with a simple button push (Figure 1d, g, h). The manual RotoPatch devices enable electricity‐free use with a simple, low‐cost design, while the motorized RotoPatch offers precise rate control and no need for user strength or dexterity. As an alternative design, we also included the eIgniter, which has fully electronic control and production of electric pulses actuated by pushbutton (Figure 1e).

In all RotoPatch designs, rotational input is converted into sequential hammer strikes via a spring‐latch mechanism, enabling consistent high‐voltage pulse generation. The manual versions use a rotary cap to facilitate repeated impacts, while the motorized version achieves programmable, repeatable actuation. The number of pulses delivered per rotation is configurable by design; prototypes featured 3 pulses/rotation (optimized for low rotational torque, suitable for motorized operation or handle‐free manual use) and 9 pulses/rotation (maximizing the pulse count per rotation for handled manual use, thereby reducing total required rotation). Increasing the pulse count per rotation necessitated steeper internal ridge geometry and consequently required higher input torque. This design flexibility highlights RotoPatch as a tunable, low‐cost, and scalable alternative to conventional electroporation systems.

For this study, electroporation systems consisted of a pulse generator (Figure 1a–e) and a MEA (Figure 1f), which comprised a 3D‐printed holder containing six rows of stainless‐steel microneedle electrodes, fabricated using a chemical etching technique. Each microneedle electrode was 50 μm thick and was arranged in a row of nine microneedles, each measuring 650 μm in length with a tip‐to‐tip spacing of 790 μm. The inter‐array spacing between adjacent electrode arrays was maintained at 650 μm. This MEA configuration enabled the electric field generated by the pulser devices to be localized within the epidermis and upper dermis, targeting skin‐resident immune cells such as Langerhans and dermal dendritic cells for enhanced vaccination.

Both piezoelectric pulsers (ePatch and RotoPatch) involve moving parts leading to noisy operation and wear and tear of mechanical parts over time. Therefore, we developed another barbeque lighter‐inspired electrical pulser, eIgniter (Figure 1e), as a fully automatic electronic pulse generator. The eIgniter consists of an ignition module (transformer and two electrodes), a 9 V battery, and a pushbutton.

We characterized the voltage profiles generated by the RotoPatch, ePatch, and eIgniter to compare their performance. In open‐circuit measurements, RotoPatch and ePatch pulsers generated bipolar oscillatory voltage waveforms with post‐pulse ringing, while the eIgniter pulser generated underdamped oscillatory waveforms (Figure 2a–c). RotoPatch generated positive and negative voltage peaks of 13.3 ± 0.3 kV (~11 μs) and −3.7 ± 0.2 kV (~22 μs), respectively (Figure 2a), which were significantly lower than those of the ePatch (24.1 ± 7.1 kV at ~11 μs and −9.0 ± 0.4 kV at ~20 μs, p <0.05, Figure 2b), and significantly higher than those of the eIgniter (16.6 ± 0.7 kV at ~4 μs and −13.6 ± 0.4 kV at ~19.3 μs, p <0.05, Figure 2c). The pulse decay times for the RotoPatch, ePatch, and eIgniter pulsers were 53.68 ± 0.47 μs, 73.93 ± 0.23 μs, and 668.03 ± 3.70 μs, respectively. The open‐circuit voltage outputs from the motorized and manual RotoPatch devices were similar (Supplementary Figure S7), indicating that the mechanism of rotational actuation did not affect the electrical performance of the motorized versus manual RotoPatch.

We also performed voltage measurements of RotoPatch, ePatch, and eIgniter administered to ex vivo porcine skin via the MEA. Compared to open‐circuit measurements, all devices showed reduced voltage amplitudes due to discharge across a closed circuit through skin (Figure 2d–f). The eIgniter produced the highest mean voltage peaks (438.8 ± 25.84 V and−335.33 ± 19.56 V, p <0.05, Figure 2f), followed by ePatch (228.20 ± 33.62 V and −262.98 ± 49.32 V, p <0.05, Figure 2e) and RotoPatch (141.98 ± 46.28 V and −160.07 ± 50.92 V, p <0.05, Figure 2d), with decay times spanning 78–132 μs. The voltage peaks observed for ePatch were consistent with previously reported measurements.45

Companion current measurements through porcine skin showed similar waveforms to the voltage outputs (Figure 2g–i). The eIgniter produced the highest peak currents (3.67 ± 0.07 A, and −2.97 ± 0.09 A, p <0.05, Figure 2i), followed by ePatch (2.78 ± 0.86 A and −2.36 ± 0.36 A, p <0.05, Figure 2h), and RotoPatch (2.02 ± 1.87 A and −2.11 ± 0.29 A, p <0.05, Figure 2g). The corresponding apparent skin impedances (i.e., the voltage divided by the current during the pulses) were 123.1 ± 7.7 Ω for eIgniter, 84.0 ± 14.5 Ω for ePatch, and 52.5 ± 18.9 Ω for RotoPatch.

Weak voltage and current signals were seen at ~300 μs when using RotoPatch (Figure 2a, d, g). We hypothesized that this was due to the hammer rebounding off the piezoelectric crystal and then striking the crystal again with lesser force before retraction. High‐speed video imaging of RotoPatch operation confirmed this hypothesis, showing a second hammer strike ~300 μs after the first one (Supplementary Video S1). While a similar second strike of the hammer was seen in video of the ePatch, we did not see evidence of it in the voltage and current profiles for ePatch (Figure 2b, e, h), perhaps because the second strike was too weak. Because there is no hammer strike involved in eIgniter operation, there would be no mechanism for this effect.

Electroporation for delivering nucleic acids such as mRNA and DNA in vaccination and therapeutic applications typically requires electric fields in the range of ~1–3 kV/cm when employing microsecond‐long pulses.30, 45 We calculated the nominal electric field strength generated by RotoPatch, ePatch, and eIgniter when pulsing pig skin using MEAs by dividing the peak voltage by the spacing between microneedle anode and cathode electrodes, which were approximately 2.8, 3.6, and 7.15 kV/cm, respectively. These values are roughly in the target range, although the eIgniter voltage was higher.

It is worth noting that the electric field strength was probably not constant throughout the treated region of skin, with increased field strength immediately next to microneedle electrode tips and edges due to their curvature and decreased field strength between microneedle electrodes of the same polarity (i.e., between microneedles in the same row).

We next compared the ability of RotoPatch, ePatch, and eIgniter to enhance delivery and expression of N1‐methylpseudouridine‐modified firefly luciferase mRNA in the skin of mice after intradermal injection of naked mRNA followed by application of 10 electric pulses (Figure 3a). Electroporation by all three devices significantly enhanced reporter protein (i.e., luciferase) expression compared to mRNA injection without electroporation (p <0.05, Figure 3b). At 24 h post‐injection, luciferase expression was boosted 2 to 4‐fold across all electroporation groups compared to the non‐electroporated mRNA group. While protein expression in the absence of electroporation rapidly declined, dropping an order of magnitude by day 5 and reaching background levels by day 9, electroporation by any of our three methods sustained significantly higher expression levels for longer durations. Electroporation by eIgniter supported expression for up to 16 days, while RotoPatch extended expression to ~21 days, and ePatch showed the most prolonged effect, with detectable expression lasting for at least 3 weeks, returning to baseline by day 30 (Figure 3b).

To quantify total expression over time, we calculated the area under the curve (AUC) of the luciferase flux profiles. The piezoelectric devices (RotoPatch and ePatch) significantly enhanced cumulative protein expression compared to no electroporation (p <0.05, Figure 3c), exhibiting a 6‐fold increase for RotoPatch and a 13‐fold increase for ePatch. Electroporation‐mediated mRNA delivery via eIgniter also resulted in a significant increase in cumulative expression relative to mRNA delivery without electroporation (p <0.05, Figure 3c). Among the three electroporation devices, there was no significant difference in their cumulative protein expression over 30 days (p >0.05, Figure 3c).

Overall, these findings highlight the potential of our multi‐pulse electroporators for carrier‐free enhancement of mRNA delivery to cells in the skin, enabling sustained protein expression.

Because electroporation is also of interest to delivering DNA into cells, we next tested the transfection capabilities of RotoPatch, ePatch, and eIgniter to deliver a GFP‐encoded plasmid DNA to cells in the skin after giving a series of 10 electric pulses (Figure 4a). Twenty‐four hours after treatment, plasmid DNA alone produced only faint GFP fluorescence, and injection of PBS alone produced background levels of signal (Figure 4b). Electroporation with the RotoPatch, ePatch, or eIgniter boosted reporter protein (i.e., GFP) expression by roughly an order of magnitude compared with the non‐electroporated plasmid site (Figure 4b). Reporter fluorescence from plasmid injection without electroporation fell to baseline within 3 days, whereas electroporated sites retained elevated expression until the end of the experiment on day 4 (Figure 4a). On day 4, the ePatch sustained the highest GFP signal, followed by the RotoPatch and, lastly, the eIgniter (p <0.05, Figure 4b). Quantitatively, day‐4 radiance dropped by 61.5% ± 12.3% for the ePatch, 55.9% ± 28.4% for the RotoPatch, and 30.4% ± 20.6% for the eIgniter relative to their respective day‐1 mean radiant efficiency (Figure 4b).

Over the 4‐day period, cumulative GFP expression (AUC) for sites electroporated by ePatch was significantly greater (~13‐fold) than at sites that received plasmid without electroporation (p <0.05; Figure 4c). The difference between GFP expression from plasmid delivery with RotoPatch (~6.6‐fold) or eIgniter (~8.5‐fold), and delivery without electroporation was not significant (p >0.05). Moreover, there was no statistical difference in GFP expression among the three electroporation devices (p >0.05; Figure 4c).

While ePatch ushered in a new approach to skin electroporation, its translation was limited by the need to apply multiple, manual thumb activations in order to administer multiple electric pulses.30, 44, 47 To address this operational limitation of ePatch, which requires ~40 N of manual normal force per pulse, we developed RotoPatch, a rotary‐actuated pulser requiring only ~5 N of tangential force. Lower force for operation, along with its ability to generate up to nine pulses per rotation from a single piezoelectric crystal, can enhance operator usability and scalability, making it better suited for routine and large‐scale use by healthcare providers requiring minimal training.

We developed three types of RotoPatches—the manual RotoPatch, manual RotoPatch with handle, and motorized RotoPatch. The manual RotoPatch is the simplest, lowest‐cost design that can administer multiple pulses with a single rotational twist of the hand, like opening the lid of a jar. While simple, this method only administered 3 pulses in this study (although future designs may be able to administer more) and required hand motion that is not easily ergonomic.

The improvement on the manual RotoPatch, we added a foldable handle to the rotation mechanism, thereby providing greater leverage when turning the rotary cap, enabling up to 9 pulses per rotation using a more ergonomic hand and arm motion. The addition of the handle increased RotoPatch size (which was minimized during storage by designing the handle to fold up) and cost, but these small negative impacts are likely to be outweighed by the positive impacts of simplified usability.

A final improvement to the RotoPatch was the use of a small, inexpensive motor to rotate the RotoPatch rotor and apply any number of pulses, according to user input. The motor resulted in a small increase in size, and likely added $2 to the cost, indicating that the motorized RotoPatch may be suitable as a fully disposable device in some markets, but may need to be reusable in low‐resource settings, in combination with a single‐use MEA component that contacts the skin.

We also evaluated a battery‐powered pulse generator with no moving parts (eIgniter) as an alternative electroporation device. The eIgniter is a more sophisticated microelectronic device compared to RotoPatch or ePatch but offers the advantage of pushbutton actuation and microelectronic control in a low‐cost format.

Among the various electroporators in this study, all of them significantly enhanced gene and protein expression following intradermal delivery of mRNA and plasmid DNA, suggesting that RotoPatch, ePatch, and eIgniter could all be useful for applications involving nucleic acid delivery. There were, however, some differences between the strength and duration of protein expression following mRNA and DNA delivery by the different electroporation devices, some of which were statistically significant and others of which were not. Additional research is needed to study transfection and expression using these devices to more fully determine not only the statistical significance of possible differences but also to assess the functional differences for specific applications.

The use of widely available piezoelectric and electrical components, such as those found in barbeque lighters, highlights the potential for rapid, global scalability of these electroporation devices for vaccination, particularly in response to future endemic or pandemic outbreak scenarios. Moreover, the prolonged expression achieved with mRNA delivered via piezoelectric electroporation highlights the promise of this technology in gene therapy applications.

While exponential‐decay and square wave pulses have traditionally been the standard for electroporation, sinusoidal bipolar oscillatory waveforms have also demonstrated efficacy in achieving reversible electroporation.27, 30, 46 Millisecond‐long pulses are often used for electroporation, but microsecond‐long oscillatory electric pulses have also been effective, attributed to sonication effects and lower membrane polarization.28, 29, 30, 31 Both piezoelectric RotoPatch and ePatch, as well as the microelectronic eIgniter, generated high‐voltage, bipolar oscillatory pulses lasting a few microseconds, although the exact waveforms of the piezoelectric pulsers and the microelectronic pulser were different. To enhance transfection efficiency, we reduced the electrode spacing to 650 μm from 900 μm previously used in ePatch,30 which resulted in a higher electric field strength in this study. Both piezoelectric electroporators generated peak electric field strengths of ~3–4 kV/cm in the skin while the eIgniter achieved peak electric field strengths up to ~7 kV/cm.

Conventional commercial electroporators employ advanced electronics to precisely control pulse parameters across microsecond to millisecond durations, which contributes to their high cost and bulky design (Table 1). In contrast, the handheld electroporators developed in this study leverage readily available components, such as piezoelectric crystals, metallic hammers, springs, and 3D‐printed plastic parts, to enable a low‐cost, scalable, and globally accessible electroporation platform.

RotoPatch, in particular, offers flexibility for diverse clinical settings: the motorized version facilitates ease of use for providers, while the manual version may be better‐suited for resource‐limited environments. Our findings demonstrate that all three electroporator platforms (RotoPatch, ePatch, and eIgniter) effectively enhance cellular uptake of nucleic acids, providing a cost‐effective and efficient alternative for gene delivery. Furthermore, the electric field strength can be adjusted by modifying the electrode spacing within the MEA, allowing customization for applications such as vaccination, gene therapy, drug delivery, and electrochemotherapy. Additionally, we introduced a battery‐powered, low‐cost electroporator (eIgniter) as an even‐simpler‐to‐use tool for nucleic acid delivery.

While RotoPatch, ePatch, and eIgniter can provide simple, low‐cost, and portable nucleic acid delivery, several limitations remain. According to their design, the voltage/electric field strength, duration, and waveform of the pulses from a given device are fixed; their modulation requires hardware changes, such as using a different piezoelectric crystal, applying a different strike force with the hammer, or altering microneedle electrode spacing. These constraints limit flexibility in pulse optimization and mean that some changes may not be possible given the limitations of pulser design. Further optimization is needed to optimize pulser performance while maintaining the simple, low‐cost device design.

The in vivo data in this study were limited to a small animal group (n = 4), focusing primarily on expression kinetics in mice and rats rather than therapeutic efficacy or immunogenicity. Future studies should evaluate vaccination potential against specific infectious diseases and the therapeutic potential of drugs enabled by electroporation via these devices in larger animal models and eventually in humans. Data were also generated by expert researchers in a laboratory environment. Future studies need to assess usability by clinicians in various healthcare settings.

Although the ePatch has previously demonstrated good tolerability in human subjects, the new device designs, the different waveform of the eIgniter, and higher electric field strengths generated by these devices (due to tighter electrode spacing in the MEA) warrant further assessment of safety and tolerability in human subjects. Future efforts should also aim to integrate drug delivery and electroporation into a single platform for one‐step delivery that avoids conventional intradermal nucleic acid injection followed by application of electric pulses using a separate device.