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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

T29 - Delivering Therapeutic Genetic Cargo Via Tattoo Gun for Direct Gene Reprogramming

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

MM

Mia Melde

Undergraduate Research Assistant
The Ohio State University, United States

Co-Author(s)

AS

Ana Isabel Salazar Puerta

Post Doctoral Scholar
The Ohio State University, United States
AG

Andrew Gotschall

MD/PhD Student
University of Miami Miller School of Medicine, United States
IO

Izabella Oliver

Undergraduate Research Assistant
The Ohio State University, United States

Primary Investigator(s)

Daniel Gallego-Perez

Professor Biomedical Engineering
The Ohio State University
Columbus, Ohio, United States

Introduction: : Type 1 diabetes mellitus (T1DM) is a metabolic disorder that affects approximately 8.4 million people worldwide [1]. In previous research, Pdx1, Ngn3, Mafa (PNM), and TCF3 genes were identified to achieve acute diabetic rescue in a model of T1DM through generation of induced pancreatic beta cells by direct reprogramming [2, 5]. The current gold standard of direct reprogramming is using viral vectors [3]; however, this method has various limitations such as limited cargo capacity, immune rejection, and toxicity. To achieve the same reprogramming efficacy that is seen using viral vectors while overcoming the prominent limitations, we propose utilizing a tattoo gun for the direct deployment of DNA encoding PNM and TCF3 into recipient tissue.
In a conventional tattoo, an immune response is triggered by the delivery of a foreign substance, such as ink, into the body [4]. Macrophages respond to the ink and engulf particles via phagocytosis, which then pass the ink onto their progeny resulting in a permanent tattoo, despite the constant turnover of epithelial cells [4]. Based on this principle, we hypothesize that a tattoo gun can effectively harness the function of macrophages, with the goal of enabling short- and long-term gene expression that facilitates potential reprogramming.

Materials and

Methods: : In these experiments, we used a commercial tattoo gun loaded with ink or phosphate buffered saline (PBS) to repeatedly penetrate the skin to aid in the subcutaneous delivery of DNA. The plasmid cocktails of interest, TCF3 or PNM, were suspended in PBS or ink at a concentration of [0.05] ug/uL per plasmid. The ink used was deionized water and witch hazel-based human-grade ink from Immortal Ink. These solutions were delivered into the subcutaneous layer of skin on the back of wildtype mice in a square of approximately 1 cm2. For the TCF3 group, different voltages (8V, 8.5V, and 9V) were also tested. Next, the optimized voltage and conditions were used for the PNM treatment. Skin was harvested from both TCF3 and PNM mice after 24 hours. qRT-PCR was used to confirm the delivery and expression of the TCF3 and PNM plasmids into the skin. Immunohistochemistry (IHC) and confocal imaging were used to determine if there was an upregulation of CD68 macrophages in the plasmid tattoos in comparison to non-tattooed skin.

Results, Conclusions, and Discussions:: The outcomes of this project demonstrate the ability to deliver therapeutic cargo into recipient tissue using a tattoo gun. After 24 hours, the expression of TCF3 was upregulated in both the PBS+TCF3 and Ink+TCF3 treated skin, with PBS+TCF3 skin having a slightly higher delivery efficacy (Figure 1A). Between each voltage, there was no significant difference in expression after 24 hours (Figures 1B,C). The next experiment focused on the cocktail of PNM, and the conditions with the highest efficacy in the TCF3 experiment were applied, being PBS as the delivery solution and a lower voltage (4V). Similarly, upregulated gene expression was found in the skin post-tattoo after 24 hours, however, only Pdx1 showed a significant amount of expression (Figures 1D,E). IHC revealed that there was an upregulation of CD68 present in the PBS+PNM tattoos in comparison to the control non-tattooed skin (Figure 1F,G), thus signifying that the tattoo successfully triggered the expected immune response. This study shows the capability of delivering plasmids into subcutaneous tissue via the novel method of tattoo gun delivery. The strategy of using tattoo penetration to directly reprogram cells presents new opportunities to achieve differentiated cells without the need of viral vectors. IHC visualized CD68 macrophages responsible for engulfing the treatments. Future efforts will use IHC to explore protein production related to T1DM reprogramming using PNM and TCF3. Since the non-tattooed skin presented very few CD68 macrophages, it became evident that the tattoo was healing properly, supporting the idea that tattooing genetic cargo will result in sustained expression. Current work is focused on further optimizing the parameters of the delivery method, such as penetration depth and the suspension media that is most effective. In future experiments, the PNM plasmids can be delivered into a T1DM therapeutic model via tattoo gun to explore the potential of acute diabetic rescue. This method could also be used for other disorders that are being treated with direct gene reprogramming. Overall, tattoo delivery proved to be a viable method for delivering plasmids encoded with factors associated with the treatment of T1DM into recipient tissue.

Acknowledgements and/or References (Optional): : [1] I. Ogrotis, T. Koufakis, and K. Kotsa, “Changes in the Global Epidemiology of Type 1 Diabetes in an Evolving Landscape of Environmental Factors: Causes, Challenges, and Opportunities,” Medicina, vol. 59, no. 4, p. 668, Mar. 2023, doi: https://doi.org/10.3390/medicina59040668.

[2] Y. Zhang et al., “PDX-1: A Promising Therapeutic Target to Reverse Diabetes,” Biomolecules, vol. 12, no. 12, p. 1785, Dec. 2022, doi: https://doi.org/10.3390/biom12121785.

[3] M. Butt et al., “Appraisal for the Potential of Viral and Nonviral Vectors in Gene Therapy: A Review,” Genes, vol. 13, no. 8, p. 1370, Jul. 2022, doi: https://doi.org/10.3390/genes13081370.

[4] A. Baranska et al., “Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal,” Journal of Experimental Medicine, vol. 215, no. 4, pp. 1115–1133, Apr. 2018, doi: https://doi.org/10.1084/jem.20171608.

[5] F. Yi, P. L. Brubaker, and T. Jin, “TCF-4 Mediates Cell Type-specific Regulation of Proglucagon Gene Expression by β-Catenin and Glycogen Synthase Kinase-3β,” Journal of Biological Chemistry, vol. 280, no. 2, pp. 1457–1464, Nov. 2004, doi: https://doi.org/10.1074/jbc.m411487200.