Combined biolistic and cell-penetrating peptide delivery for an optimised intradermal DNA delivery platform
Publication Date
December 12, 2025
Creator
Abstract
Physical gene delivery via biolistic methods involves the precipitation of nucleic acids onto carrier particles and their direct transfection into exposed tissues (e.g. skin) by high-velocity propulsion, such as with the Helios gene gun system. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system utilises fusion peptides composed of cell-binding, nucleic acid-condensing, and cell-penetrating domains to enhance transfection across multiple cell types. In this study, chemical (GET) and physical (gene gun) DNA delivery systems were combined under the hypothesis that this integration may enhance cellular distribution and uptake in regions not directly transfected by biolistic penetration.
The proposed biolistic delivery platform was evaluated using several gene bullet variants. Three main prototypes namely GET-gold 1µm, GET-iron 500nm, and GET-iron 1µm, were selected after screening over one hundred candidate formulations. These were incorporated into gene gun-compatible dry bullets via lyophilisation, offering a simple and scalable alternative to conventional methods. The bullets were subjected to a comprehensive series of quality assessments, including formulation and bullet production optimisation, physicochemical characterisation, DNA quantification and complexation analyses, in vitro transfection and cell viability assays, preliminary in vivo reporter gene expression, and a proof-of-concept vaccination study. Gold spermidine bullets served as the benchmark for comparison.
The first prototype, GET-gold 1µm bullet, demonstrated enhanced reporter gene expression in vitro in monolayer cultures and engineered tissue, while maintaining cell viability. Both flushed and fired GET-gold1µm bullets retained significant enhancement of chemical- and physical-mediated transfection in vitro. A SARS-CoV-2 plasmid DNA vaccine encoding the spike receptor-binding domain (S-RBD) was delivered to mice using these bullets. While specific T cell and antibody responses were generated, further research is required to determine the magnitude of these immune responses between different delivery systems. Limited DNA loading and overly tight complex binding were identified as
key limitations of this prototype.
To address these limitations, functionalised dextran-coated iron oxide nanoparticles were explored as alternative carriers. The resulting GET-iron 500nm bullets exhibited excellent DNA loading through an electrostatic binding mechanism. This enabled nearly complete DNA complexation, following optimisation of loading conditions and particle characteristics. Although enhanced transfection was observed in vitro, preliminary in vivo reporter gene expression remained suboptimal. The poor physical-mediated transfection was attributed to the reduced size of the iron carrier, which was half that of the gold carrier used in spermidine bullets.
A final prototype, GET-iron 1µm bullet, was generated using a custom-ordered dextran-coated, carboxylated, 1 µm, iron oxide carrier. Despite achieving significant in vitro chemical- and physical-mediated transfection, in vivo gene
expression remained low. The core-shell structure, irregular cluster shape, and low density of the iron1µm particles likely contributed to insufficient momentum, velocity, and tissue penetration, thereby limiting delivery efficacy. A denser, more spherical, and uniform carrier is likely needed to improve projectile kinetics.
In summary, the combined GET and gene gun delivery approach showed potential as an alternative DNA delivery platform. However, further refinement of carrier particle properties and formulation parameters is required to achieve a scalable and effective intradermal gene delivery system suitable for nucleic acid vaccination.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Pharmacy (UK)
eprints ID
82721
UoN Repository URI
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So, Roizza, 20250839, final version.pdf
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Full-text
Description
Examined
Size
15.31 MB
Format
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