Poster W14 - DNA Transfection by Cutaneous Suction Application shows Correlation with Tissue Strain and Tissue Tension

Introduction: In vivo transfection has many therapeutic applications such as nucleic-acid-based vaccination, gene therapy for neurodegenerative disorders, AAV (adeno-associated virus) cloning for clinical therapeutics, and AeTEP-1 transfection for reducing dengue infection, etc. Among various methods of transfection, DNA transfection by physical methods are becoming more popular in order to avoid the potential adverse effects by chemical or biological agents. A recently developed physical method of DNA transfection has been demonstrated using cutaneous suction application, which is easy to use, painless, and cost-effective. The method uses naked plasmids that avoids adjuvant toxicity or other potential adverse effects of chemical transfection. Suction-based DNA vaccination has shown similar B cell responses but greater T cell responses for a COVID-19 vaccine when compared to other delivery methods in both pre-clinical and clinical trials.

Materials and

Methods: A parametric study was conducted on the effectiveness of cutaneous suction-based DNA transfection. Green Fluorescent Protein (GFP) plasmid is used as a transfection reporter in a rat model. A 25ug of pEGFP-N1 in 50ul solution is injected on the rat dorsal skin using Mantoux injection technique. Following the injection, suction application is performed varying suction pressure (35 – 90 kPa) and diameter of the suction-nozzle (2 – 8 mm). The GFP expression was quantified after 24 hours of injection and suction application. The ‘Total Pixel Count’ and the ‘Total Intensity’ of the GFP expression from an image is calculated as the total number of non-zero and non-negative pixels and the sum of the intensities of all non-zero and non-negative pixels, respectively. Normalized values are obtained by dividing Total Intensity or Total Pixel Count by the respective standardized control value (pEGFP-N1 injection only without suction application).

Numerical modeling is performed with a 2D axisymmetric model where negative pressure is applied onto tissue. The thickness and mechanical properties of the tissue layers are obtained from histopathological images of rat skin and from the literature, respectively. Negative pressure loads are applied to the tissue top surface within the nozzle boundary, where slip is allowed between nozzle and tissue surface. The Frobenius norm of the strain tensor is computed at different negative pressures and nozzle sizes. The integrated total strain (ITS) is calculated from the Frobenius norm of strain tensor above a strain threshold at the corresponding volume of integration for each case.

Results, Conclusions, and Discussions: The suction application needs to be applied on skin following the pEGFP-N1 injection as suction before injection generates a much lower GFP expression compared to the case of suction after injection. In cases of pEGFP-N1 injection followed by suction application, the GFP expression area and pattern is found to be correlated with nozzle size and shape. The expression shapes are approximately circular following the circular nozzles and the expression areas are same as the nozzle area, with the exception of the 8 mm nozzle due to its size being bigger than the injection bleb (DNA cargo). The strain patterns from numerical modeling strongly matches with the GFP expression patterns. For 6 mm and 8 mm nozzles, the strain in tissue is stronger towards the nozzle rim, while the strain is similar at the suction rim and the aspirated tissue dome for 2 mm and 4 mm nozzles. Similarly, the GFP expression is more uniform for the 2 mm and 4 mm nozzles, whereas the expression is greater towards the rim for 6 mm and 8 mm ones. While investigating the suction pressure variation, the 35 kPa case shows similar or less GFP expression than the control case. However, the GFP expression shows a positive correlation with both nozzle size and suction pressure from the threshold suction pressure ~50 kPa. Moreover, the GFP expression is found to be correlated to the integrated total strain (ITS) above a strain threshold of 0.15 from the numerical modeling and tissue tension which is a product of pressure and circular nozzle diameter, excluding three cases of 35 kPa (below threshold suction pressure).

This work shows a parametric study of suction-based transfection, including nozzle diameter and suction pressure, where the GFP expression is found to be dependent on both parameters. The quantified GFP expression level shows a positive correlation with the integrated total strain above a strain threshold of 0.15 from the numerical modeling. Additionally, the GFP expression is strongly correlated to the tissue tension for the cases from the threshold suction pressure (50 kPa), which is a simple-to-use parameter for circular nozzles.

Acknowledgements (Optional): The authors acknowledge funding support from GeneOne Life Science, Inc. (Seoul, South Korea).