
==== Front
STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00426-X
10.1016/j.xpro.2024.103261
103261
Protocol
Protocol for in vivo nucleic acid delivery utilizing the rolling microneedle electrode array
Wu Pengfei 15
Yang Tongren 12
Huang Dong 23
Zhang Tian 1
Naeem Abid 1
Ren Yingjie 3
Wang Yushu 4
Li Zhihong 3
Huang Yuanyu yyhuang@bit.edu.cn
1∗
Li Yong liyong2021@bit.edu.cn
1∗∗
Weng Yuhua wengyh@bit.edu.cn
156∗∗∗
1 School of Life Science, Advanced Research Institute of Multidisciplinary Science, Key Laboratory of Molecular Medicine and Biotherapy, Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Beijing Institute of Technology, Beijing 100081, China
2 National Key Laboratory of Advanced Micro and Nano Manufacture Technology, School of Integrated Circuits, Peking University, Beijing 100871, China
3 AciMicro Medical Technology, Guangzhou 510700, China
4 Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA
∗ Corresponding author yyhuang@bit.edu.cn
∗∗ Corresponding author liyong2021@bit.edu.cn
∗∗∗ Corresponding author wengyh@bit.edu.cn
5 Technical contact

6 Lead contact

13 8 2024
20 9 2024
13 8 2024
5 3 103261© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Electroporation temporarily enhances cell membrane permeability and promotes the absorption of external molecules. We have developed a device termed the rolling microneedle electrode array (RoMEA) that combines a densely arranged microneedle array of electrodes with rolling structures. Use RoMEA to create uniform skin micropores for efficient, low-damage transfection of nucleic acids over extended areas of the body. We describe in detail the design, fabrication, and assembly of the device and the application of in vivo electroporation of nucleic acids.

For complete details on the use and execution of this protocol, please refer to Tongren Yang et al. 1

Graphical abstract

Highlights

• Detailed description of the design of RoMEA

• Steps for assembling the microneedle electrode array of RoMEA

• Procedure for in vivo nucleic acid electroporation using RoMEA

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Electroporation temporarily enhances cell membrane permeability and promotes the absorption of external molecules. We have developed a device termed the rolling microneedle electrode array (RoMEA) that combines a densely arranged microneedle array of electrodes with rolling structures. Use RoMEA to create uniform skin micropores for efficient, low-damage transfection of nucleic acids over extended areas of the body. We describe in detail the design, fabrication, and assembly of the device and the application of in vivo electroporation of nucleic acids.

Subject areas

biophysics
molecular biology
biotechnology and bioengineering
==== Body
pmcBefore you begin

Electroporation is a physical method for directly transfecting nucleic acids into cells and tissues. During this process, the applied electric field generates temporary micropores in the cell membrane, increasing its permeability and allowing exogenous nucleic acids to pass through these micropores into the cytoplasm, where they can function.2,3,4,5,6,7,8,9 After electroporation, cells remain in an unstable state for several minutes before the membrane defects are automatically repaired.10 This electric pulse-based electroporation method has successfully transfected various nucleic acids into different cells.11,12 As a non-viral nucleic acid delivery technology, electroporation avoids the immunogenicity and toxic side effects associated with delivery vectors, achieving efficient nucleic acid transfection. Other advantages of this technology include no significant limitation on the size of the transfected nucleic acid molecules, making it suitable for delivering both small nucleic acids and larger ones such as plasmid DNA and mRNA. Additionally, electroporation equipment is reusable, structurally simple, easy to produce, and cost-effective. Numerous studies have also confirmed the safety of electroporation in animal and human experiments.10,13,14,15

Despite the promising applications of electroporation, achieving efficient nucleic acid transfection in large tissue areas remains challenging. The main factors affecting electroporation efficiency include electric field strength, pulse width, shape, frequency, duration, and electrode design.16,17 To achieve efficient and safe electroporation in large tissue areas, it is necessary to consider these conventional factors and also to design equipment that can accommodate the electroporation of large tissue areas and ensure safety. Only by comprehensively considering these factors can the goal of efficient and safe electroporation of large tissue areas be achieved.

We have developed a novel Rolling Microneedle Electrode Array (RoMEA) device.1 This portable device combines electroporation technology with rolling microneedle arrays, utilizing parallel circular blades and edge microneedle arrays as electrodes, which are arranged at close intervals to create regular, painless micropores on the tissue surface, enabling efficient, low-damage nucleic acid transfection over large tissue areas. Our experimental results demonstrate that RoMEA can achieve efficient delivery of siRNA in large areas of mouse muscle tissue, effectively promoting gene silencing in mice. This suggests that RoMEA can serve as a safe and efficient nucleic acid delivery platform technology for disease treatment and medical cosmetics. This protocol mainly describes four key steps in the application of RoMEA technology: (1) RoMEA design, (2) Microneedle array electrode blade manufacturing, (3) RoMEA assembly, and (4) RoMEA electroporation process.

Institutional permission

We used female C57BL/6 mice aged 6–8 weeks. All experiments were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee, and must comply with state and institutional regulations. This protocol’s experimental procedures have been certified by AAALAC as being conducted in a Specific Pathogen Free (SPF) laboratory animal facility. Additionally, all experimental animals were treated in accordance with the program approved by the Animal Protection and Use Committee of Peking University.

RoMEA design

Timing: 3–5 days

RoMEA device integrates the advantages of a roller and a microneedle electrode array. Through precisely controlling the exposure length and spacing of the microneedle electrodes, RoMEA penetrates deep into the skin and subcutaneous tissue while rolling, achieving gentle, consistent electroporation.

The device is primarily composed of blades, spacers, metal wires, a handle, slip rings and insulating caps (Figure 1A). The blades serving as the microneedle array electrodes are made of 316 stainless-steel with a thickness of 100 μm and a diameter of 21 mm. Each blade is designed with 60 microneedles, a large round hole and another smaller one. The length of the microneedles is approximately 2 mm, but we only expose a quarter of that length (500 μm) as an electrode. This design controls the penetration depth. The angle difference between the adjacent microneedle tip and the center is 6°, and the spacing is 1 mm. When adjacent metal sheets are installed, the angle difference is 3°, resulting in alternating arrangement of positive and negative microneedle tips. The distance between the adjacent electrodes is 700 μm, which can effectively penetrate the high-resistance stratum corneum, generate a uniform electric field within the tissue, and facilitate rolling and electroporation on the target tissue.Optional: In this design, we used an optimized 0.5 μm length microneedle tip for electroporation. This depth causes minimal damage to the skin and is almost painless, minimizing the risk of physical trauma and infection. The overall length of the microneedle is adjustable, and the exposed microneedle length can be monitored by adjusting the diameter of the spacer, such as by adjusting the spacer diameter to be 1 mm or 1.5 mm, with a maximum length of 2 mm less than the diameter of the microneedle blades to expose 500 μm, 750 μm, 1 mm of the microneedles as electrodes for electroporation, respectively.

Note: The longer the microneedles, the greater the potential for skin damage. The stainless-steel microneedles with a thickness of 100 μm possess good strength and will not bend after penetrating the skin. This helps avoid local short circuits and electrical sparks during electroporation.

Figure 1 The structural design of the Rolling Microneedle Electrode Array

(A) The overall design of RoMEA device.

(B) Illustration of the continuous electrode array.

Spacers are 500 μm thick and approximately 20 mm in diameter and are used to prevent short circuits between the positive and negative electrode arrays and to maintain an appropriate distance (Figure 1B). They also have two round holes, corresponding to the holes in the blades. Metal wires are used to connect the positive and negative blades and are connected to the electroporation apparatus. The insulated caps are employed to secure the microneedle blades and spacers. The slip rings enable the microneedle array to roll continuously and the handle is easy to operate.

The area of the electrode head contacting the target tissue is approximately 1.5 cm × 1 cm, and the electroporation can cover an area of 1.5 cm square without rolling.Optional: For larger animals, such as rabbits or pigs, perform in vivo electroporation on the target area by rolling the electrode head multiple times.

Note: The RoMEA device is designed using SolidWorks software. We have successfully manufactured and repeatedly tested this device. Additionally, parameters such as the length of the microneedle electrodes and the distance between adjacent electrodes can be modified according to specific experimental purposes.

Microneedle array electrode blade manufacturing

Timing: 2–3 days

The blades of the RoMEA microneedle electrode array are fabricated through photolithography and wet etching. Photolithography is a process similar to photographic reproduction in which a pattern is precisely transferred from a mask to a photoresist (a light-sensitive coating) applied to the surface of a stainless-steel plate. This is followed by processes such as ion implantation, etching, and thin-film deposition under the protection of the photoresist (Figure 2).1. Wash the stainless-steel plate with ethanol and deionized water 5 times, and then dry it naturally.

Note: This step enhances the adhesion of the photoresist to the substrate.

Note: The surface area of the stainless-steel should be slightly larger than the area required for the microneedle array roller.

Note: The selected stainless-steel is 316 type. This grade was chosen for its enhanced corrosion resistance, high temperature strength due to the addition of Mo, excellent work hardening properties (non-magnetic), high temperature resilience and good surface gloss and aesthetics of cold-rolled products.

2. Apply the photoresist to the surface of the stainless-steel by rotating. The photoresist is dropped on the stainless-steel surface and rotated at a speed of 1000–1600 g to form a uniform coating on the entire surface.

Note: Photoresist is a type of polymer that is sensitive to light (or electron energy) and is used as a medium to transmit ultraviolet light or electron beam exposure mode. It acts as an etch-resistant layer to protect the substrate surface.

3. Create a photoresist mask by using double-sided photolithography. By exposing the desired pattern area on the photomask to a specialized light source, a photochemical reaction is induced in the photoresist in that area, aiming to transfer the mask pattern to a photoresist coated stainless-steel plate.

4. Develop the photoresist. Dissolve the soluble parts of the photoresist using a developer solution to form the pattern.

5. Wet-etch the stainless-steel plate with a corrosive liquid.

6. Remove the photoresist and rinse the microneedle array roller with ethanol and deionized water 5–8 times. Examine the pattern of the photoresist to assess the quality of the photolithography.

CRITICAL: This detailed protocol outlines the precise steps involved in creating the microneedle array electrode blades for RoMEA, emphasizing the importance of each stage to achieve the desired pattern and quality.

Note: The two round holes in the blades were also manufactured in the same way. Other parts of electroperforation such as handle, slip ring and spacer are manufactured in the factory.

Figure 2 Fabrication Process Flowchart for Microneedle Array Electrode Blades

RoMEA assembly

Timing: 2–4 h

From the structural design, it is evident that the microneedle electrode array of RoMEA is a stacked structure of "positive electrode - spacer - negative electrode - spacer". The specified positive electrode blades are connected via a metal wire, while the negative electrode blades are connected by another metal wire. The roller handle is made of polyvinyl chloride (PVC) material, and the rotating shaft is a rolling structure. The microneedle array and spacers are installed around the rotating shaft. In RoMEA, the distance between the positive and negative electrodes is short, making it crucial to connect adjacent blades to different polarities. Hidden metal wires are connected to small holes in the blades through adjacent spacers and large holes in the blades to generate sufficient electric fields at low voltages. This specific method of parallel and separate connections effectively isolates each blade from the others. As a result, damage caused by microacupuncture into tissues and burns caused by RoMEA electroporation can be significantly reduced or even eliminated. The edge microneedle arrays serve as electrodes, preventing the positive and negative electrodes from connecting. The metal wires are sequentially connected as a microneedle array serving as anodes, with the cathode avoiding contact with them. This design ensures effective electroporation and minimizes potential tissue damage.7. Assembly of roller microneedle array.a. Begin by installing the roller microneedle array as illustrated in Figure 3C.

b. When assembled, layer the components from the outside to the inside in the following order: insulation cap, microneedle array, spacer, and then another microneedle array.i. As in Figure 3A, thread a wire extending from a positive conductive ring through a small hole in the red metal plate, and weld it securely using stainless-steel flux and solder (red dotted line).

ii. Pass the wire through a hole in a spacer and a larger hole on a black stainless-steel plate,

Caution: ensuring that the positive electrode is connected and does not come into contact with the black metal plate.

iii. Weld the wire through the spacing thread to a small hole in the second red stainless-steel plate.Note: Applying the same procedure to a black metal plate connected to the negative end (black dotted line) allows the connection and isolation of positive and negative microneedles across the entire electrode head, thus guaranteeing that adjacent rows of microneedle tips have opposite polarities.

Note: The spacer plays a crucial role in providing insulation. This specific forked connection design enables isolation of each blade from the others. During the assembly process, use a multimeter to check if the connections are correct.

Caution: The blades are sharp. Care should be taken to avoid injury during assembly.

8. Securing the Slip Rings.a. Affix the slip rings to both ends of the insulation cap, matching them to the groove on the front of the handle where the arrow symbol is marked in Figure 3B.

Note: Ensure this step to maintain the structural integrity and functionality of the array.

9. The inside of the handle has a "Y"-shaped groove, which is designed to embed the metal wires as shown in Figure 3B.a. The two metal wires of the assembled electrode emerge from the head of the "Y" shape and then converge at the tail of the "Y".

b. Connecting the wires (green circle) to the electroporation instrument establishes the electrical pathway.

10. Assembly of the Handle.a. Compose the handle using two parts as shown in Figure 3B.i. One half of the handle has a characteristic groove (red circle), while the other half has a corresponding bulge (blue circle).

ii. During installation, align the groove with the bulge and fit them together.

Figure 3 Installation of RoMEA

(A) Installation Diagram of Electrode Array.

(B) Installation diagram of slip rings and handles.

(C) Installation diagram.

(D) Finished RoMEA product.

View Figure 3D to observe the assembled ROMEA.Note: Use a multimeter in the final connection process to ensure that all connections are correctly made.

CRITICAL: These steps outline a careful and methodical approach to assemble the RoMEA device, emphasizing safety and accuracy to ensure proper operation and minimize risks during use. Generally, reuse ROMEA after disinfecting it with ultraviolet light or spraying it with 75% alcohol, until it ceases to function or debris on the needle tip cannot be removed.

Table 1 Electroporation settings

Parameter	Setting	
Pulse Voltage	50 V	
Number of Pulses	10 times	
Pulse Interval	1 s	
Pulse Duration	10 ms	

Figure 4 Electroporation program of RoMEA

(A) Anesthetized state of the mouse.

(B) Performing electroporation.

(C) Post electroporation.

(D) Electroporation device.

(E) An electroporation device connected to a wire.

(F) Prepare wires for connection to RoMEA.

(G and H) Parameter settings during electroporation.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
Photoresist	Microflu Microfluidics Technology (Changzhou) Co., Ltd.	AZ 4533	
Tetramethylammonium hydroxide	Tansoole	75-59-2	
Hydrochloric acid	Tansoole	7647-01-0	
Ferric chloride	Tansoole	7705-08-0	
	
Experimental models: Organisms/strains	
	
Mice: C57BL/6 (female mice aged 6–8 weeks)	Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China)	N/A	
	
Software and algorithms	
	
SolidWorks (version 2018)	SolidWorks	https://www.solidworks.com/; RRID: SCR_024908	
GraphPad Prism (version 8.0)	La Jolla, CA, USA	https://www.graphpad.com/; RRID:SCR_002798	
Molecular imaging soft package	Carestream Health (American)	N/A	
	
Others	
	
Red fluorescent protein (pmRFP-C1) plasmid	Addgene	# 54764	
Cy5 labeled siRNA	Suzhou Ribo Life Science, Co. Ltd. (Kunshan, China)	N/A	
Isoflurane	RWD Life Science Co., Ltd. (Shenzhen, China)	R510-22-10	
Syringe	Becton Dickinson and Company (American)	320310 1mL U100	
Depilatory cream	Purchased online from Jing Dong (Beijing, China)	N/A	
Electric soldering iron	Purchased online from Jing Dong (Beijing, China)	N/A	
Slip ring	BSPD (Shanghai) bearing manufacturing Co., Ltd.	681XZZ	
Spacer	Shanghai Lunyi Acrylic Products Co., Ltd.	N/A	

Materials and equipment

Hyaluronidase solution

Reagent	Final concentration	Amount	
Hyaluronidase	2 μg/μL	40 μg	
PBS	0.01 M	2 μL	
Total	N/A	20 μL	

Note: Hyaluronidase can be prepared in advance and can be stored for up to 2 years at −20°C.

Cy5-siRNA solution

Reagent	Final concentration	Amount	
Cy5-siRNA	1.0 μg/μL	20 μg	
Diethyl pyrocarbonate (DEPC) treated water	N/A	20 μL	
Total	N/A	20 μL	

Note: The siRNA stock solution can be stored for up to 1 year at −20°C or −80°C.

Alternatives: The working concentration of cy5-siRNA solution can be 0.5 μg/μL, or can be selected according to needs.

RFP plasmid solution

Reagent	Final concentration	Amount	
RFP plasmid	0.5 μg/μL	10 μg	
DEPC treated water	N/A	20 μL	
Total	N/A	20 μL	

Note: The RFP plasmid can be stored for up to 1 year at −20°C or 2 years at −80°C.

Alternatives: We can select plasmid according to needs, such as Green Fluorescent Protein (GFP) plasmid.

Step-by-step method details

Electroporation program of RoMEA

Timing: 2–4 h

1. Preparation for electroporation.a. Before electroporation use the pet shaver and depilatory cream to remove the hair on the rear buttocks of the mice.Note: When applying the depilatory cream, the time should be controlled at about 2–3 min.

CRITICAL: If the time is too long, the depilatory cream may irritate or damage the skin and affect the subsequent experiment.

b. Sterilize the RoMEA equipment with 75% medical alcohol and dry before use.

Electroporation protocol

Timing: 2–3 h

2. Use isoflurane to anesthetize mice first (Figure 4A).

3. Injection of hyaluronidase solution.a. Take 20 μL of hyaluronidase with a concentration of 2 μg/μL and inject it into the skin, about a depth of several hundred microns to 1 mm of the skin.

b. Leave it for about 15 min to loosen muscle tissue.

Note: The function of hyaluronidase is to loosen the extracellular matrix, significantly enhancing the diffusion of nucleic acids.

4. After 15 min of enzymolysis, inject 20 μL of nucleic acid solution subcutaneously at the same site where the hyaluronidase was injected.

Note: The nucleic acid can be plasmid, ASO, siRNA, mRNA or other kinds. We recommend a nucleic acid concentration of 500 ng/μL or 1000 ng/μL.

5. Before proceeding, connect the electroporation device as shown in Figures 4D–4H.a. Attach the BTX ECM830 square wave electroporation system via the gold wires (anode and cathode) of the handle.

b. Set the relevant electroporation parameters as detailed in Table 1.

Note: The pulse voltage used in the experiment is the optimized voltage obtained from the experiment. When exploring the voltage, the pulse voltage used is 30 V, 50 V, 70 V and 90 V. The rest of the electroporation parameters are fixed and can be changed slightly according to the specific experiment.

6. Electroporation in vivo.a. Place the RoMEA gently on the surface of the target skin.

b. Activate the electroporation device, and roll it back and forth across the skin 3–5 times.

7. Move the roller to the adjacent area and repeat the pulse stimulation until the entire area has been treated as shown in Figure 4B.

CRITICAL: If a slight tremor is observed in the mice, it indicates mild pain. This can be avoided by adjusting the parameters, e.g. by lowering the voltage.

Post-electroporation procedure

Timing: 2–3 h

8. After completing electroporation, remove RoMEA from the body, and put the mice back in the cage, then observe the mice (Figure 4C).

Utilize in vivo imaging systems to examine the RFP signals in the thigh muscles of all mice at specific times post-electroporation. The following are the detailed procedures for in vivo fluorescence imaging.Note: Determine the detection time based on the specific objectives of the experiment, and continuously monitor signals at different time points.

a. To evaluate the electroporation effects of RoMEA, observe the mice using a fluorescence in vivo imaging system (Carestream In-Vivo Imaging System FX Pro, Carestream Health, USA, Bruker Corporation). In this experiment, use 550 nm excitation and 600 nm emission filters for RFP plasmid imaging, and 630 nm excitation and 700 nm emission filters for Cy5-siRNA imaging.

b. Anesthetize the mice with a mixture of isoflurane and oxygen using an evaporator (Matrix VIP3000 Isoflurane Evaporator, Midmark Corporation, Ohio, USA) before and during imaging.

c. Record fluorescence intensities using a small animal imager.Pause point: Imaging of mice in different groups should be performed at the same time intervals as much as possible to ensure the consistency of experimental time.

d. Quantitatively analyze the collected fluorescence intensities using software (Carestream Health, USA).Note: If hair is present at the electroporation site before imaging, remove it in advance with hair removal cream to facilitate observation. In addition, to ensure the accuracy of the experiment, set at least three replicates for each experimental group.

Expected outcomes

This study investigated whether RoMEA can promote transdermal delivery of nucleic acids. In order to optimize the optimal electroporation voltage parameters, different voltage conditions were set up to deliver the plasmid of red fluorescent protein (RFP), including PBS group with 0 V, plasmids groups with 30 V, 50 V, 70 V and 90 V.

The results show no significant protein expression at 0 V and 30 V. However, noticeable protein expression was observed at 50 V, 70 V, and 90 V (Figure 5A), with fluorescence intensity peaking at 50 V (Figure 5B).Figure 5 Whole-Body Fluorescence Imaging of Mice Post-Electroporation with RFP Plasmid Using RoMEA

(A) Injection of RFP plasmid into the shaved thighs of normal mice, followed by electroporation and monitoring at specified time points.

(B) Quantitative analysis of (A). Reprinted with permission from Tongren Yang et al. (2021).1

RoMEA’s efficiency and transfection duration were also assessed using Cy5-siRNA as the transfection agent over 1, 2, 3, 4, and 5 days, with voltages set at 0 V, 30 V, 50 V, and 70 V. The results indicated that at 0 V, the fluorescence signals in the mice’s legs gradually disappeared after 2 and 3 days. In contrast, animals treated with RoMEA at 30 V, 50 V, and 70 V showed prolonged Cy5 presence in the target tissues (Figure 6A). This confirms that siRNA was delivered into deep tissues and remained there for a long time. The strongest signal and longest duration were observed in groups treated at 50 V (Figure 6B).Figure 6 Whole-Body Fluorescence Imaging of Mice Post-Electroporation with Cy5-siRNA Using RoMEA

(A) Injection of Cy5-siRNA into the shaved thighs of normal mice, followed by electroporation and monitoring at specified time points.

(B) Quantitative analysis of (A). Reprinted with permission from Tongren Yang et al. (2021).1

The risk of electrical burns or penetration injuries directly impacts electroporation devices’ clinical application, affecting therapeutic outcomes and subsequent procedures. Therefore, RoMEA’s safety was further evaluated. In order to evaluate whether microneedle penetration and electric pulse stimulation would cause damage, we observed the appearance of the skin and tissue sections before and after treatment with electric pulses (voltage: 50 V, duration 10 ms, interval 1 s, 10 pulses) under a microscope.

The results demonstrated that after electroporation, only fine pores were observable on the skin, which completely recovered within a few days (Figure 7A). Those observations were further supported by histopathological staining analysis (Figure 7B).Figure 7 Safety Assessment of the RoMEA Platform

(A) Observation of the skin appearance before and after electroporation with RoMEA.

(B) H&E staining of skin tissues subjected to electroporation with 50 V at specific recovery time points. Scale bar: 200 μm. Reprinted with permission from Tongren Yang et al. (2021).1

Limitations

Electroporation technology uses high-voltage pulses to temporarily increase cell membrane permeability, enhancing the absorption of therapeutic substances such as drugs or nucleic acids.18 The technique is particularly suitable for treating skin diseases, where local electroporation delivery significantly increases the concentration of the drug in the target cells while reducing the impact on surrounding normal tissue, minimizing therapeutic toxicity.

However, the application of electroporation in deep tissue lesions (such as most solid tumors) faces inherent limitations. On the one hand, deep tumors are embedded in tissue, and electroporation risks compromising the integrity and function of neighboring healthy cells, limiting its applicability as a local treatment for deep tumors.

Another possible specific challenge associated with RoMEA is that it is sometimes not possible to ensure effective delivery across various organization types. Different tissues show different degrees of resistance and thickness, which affects the performance of microneedles and the electroporation process. Controlling the penetration depth is crucial; too deep penetration can cause unexpected damage or discomfort, while insufficient penetration can result in ineffective nucleic acid delivery. This limitation requires precise calibration of the device for different applications, which may limit its use in certain tissues or specific treatments.

Finally, the application of ROMEA is not a single step. The therapeutic agent is first injected into the target area using a syringe, followed by electroporation with RoMEA. In addition, this method requires the use of specialized equipment and predetermined parameters. Optimizing the electrical pulse voltage is essential for maintaining cell viability, effectively addressing the safety concerns associated with this process.

Troubleshooting

Problem 1

RoMEA does not work (related to RoMEA assembly of Step 3 before you begin).

Potential solution

Before installing RoMEA, ensure that your hands and all tools are dry, as any moisture might cause accidental short circuits. Then, proceed with a multimeter check. Set the multimeter to resistance test mode. Using its probes, touch the positive and negative electrode blades separately. If the multimeter emits a beep or shows a low resistance value, it could indicate a potential short circuit between the blades. Based on the multimeter’s feedback, adjust the positions of the blades and metal wires until you confirm the absence of any short circuit. After reassembling and making adjustments, use the multimeter once more to verify that everything is correctly set up and free of problems.

Problem 2

The fluorescence signals observed in the mice’s legs exhibited uneven distribution (related to step 3 in step-by-step method details).

Potential solution

During the electroporation process, ensuring the accuracy and correct sequence of each step is crucial. Particularly, after completing the first electroporation cycle, it’s essential to relocate the roller to a new position before initiating the subsequent cycle. Electroporation while moving the roller may result in inconsistent nucleic acid delivery into the target cells. This inconsistency can adversely affect the treatment’s effectiveness and uniformity. Hence, it is imperative to ensure that the roller has been completely moved to its new position before commencing the next electroporation cycle.

To optimize the outcomes of electroporation and maintain consistent treatment quality, operators must receive proper training and adhere strictly to the operating procedures. Furthermore, utilizing high-quality instruments and equipment, complemented by regular maintenance and calibration, is key to ensuring the accuracy and reliability of the electroporation process.

Problem 3

Improper installation or lack of safe component compatibility.

Potential solution

The meticulous design of the electroporation setup is vital for ensuring smooth operation. It’s crucial to verify that each component is correctly sized and installed, as improper installation or mismatched components can result in equipment malfunction or damage. Such issues can disrupt the treatment process and potentially introduce safety hazards. To address this, resizing and precise calibration of parts are recommended. Concurrently, adjusting the device’s parameters to align with the specific needs of the experiment is also essential for optimal performance.

Problem 4

Photoresist shows small islands or pinhole-like spots (related to the manufacturing of microneedle array electrode blades).

Potential solution

Poor pre-coating cleaning of the substrate surface, which is contaminated with dirt and micro-fine dust particles, can impede both exposure and etching processes where dust exists within or on the surface of the photoresist layer. Therefore, it is crucial to ensure that the substrate surface is thoroughly cleaned before the coating process.

Problem 5

During the electroporation process, the RoMEA device stops working (related to electroporation protocol in step-by-step method details).

Potential solution

In the Electroporation Protocol, one way to determine if the electroporation is functioning correctly is to observe the intended number of pulses. For example, if 10 pulses are set, you should feel 10 distinct pulses and notice physiological twitching in the mouse. If fewer than the set number of pulses are detected, check if the connection between the electroporator and the RoMEA device has become disconnected. Disconnect the power, reconnect the equipment, and repeat the experiment.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yuhua Weng (wengyh@bit.edu.cn).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be fulfilled by the technical contact, Yuhua Weng (wengyh@bit.edu.cn).

Materials availability

Plasmids generated in this study have been deposited to Addgene, mRFP1-C1 and plasmid # 54764.

Date and code availability

This study did not generate/analyze datasets/code.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2021YFC2302400 and 2021YFA1201000 ), the National Natural Science Foundation of China (32171394 ), the Beijing Nova Program (Interdisciplinary Cooperation Project) from Beijing Municipal Science and Technology Commission (20220484207 ), and the Fundamental Research Funds for the Central Universities (2022CX01013 ). We thank the Biological and Medical Engineering Core Facilities and the Analysis and Testing Center, Beijing Institute of Technology for supporting experimental equipment and the staff for valuable help with technical support.

Author contributions

P.W. conceived and performed the experiments and wrote the manuscript. T.Y. was involved in discussion and literature reviewing. D.H. and T.Z. aided in crafting the manuscript and contributed with their subject matter knowledge. A.N., Y.R., Y. Wang, and Z.L. reviewed the manuscript and provided critical comments. Y.L. supplied valuable advice. Y. Weng. and Y.H. supervised the project, reviewed the manuscript, and provided financial support.

Declaration of interests

The authors declare no competing interests.
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