ORCID
https://orcid.org/0000-0003-3175-2427
Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
David Punihaole
Abstract
Efficient delivery vectors are essential for the success of gene therapies, which hold the potential to treat a wide range of challenging and rare diseases. While viral vectors remain the most widely used delivery systems, they suffer from immunogenicity and high production costs. In contrast, cationic polymers, such as polyetheleneimine (PEI)-based systems offer a safer and more accessible alternative, but their clinical translation has been hindered by lower delivery efficiencies.
Improving the performance of PEI-based systems requires a deeper understanding of how PEI binds to DNA and how this interaction affects DNA release inside cells. PEI systems must be carefully tuned to protect DNA during delivery, while still being able to release it effectively at the right time and place inside the cell. PEI-DNA complexes are commonly believed to form through electrostatic interactions between the positively charged PEI and negatively charged DNA. However, the details of this interaction are still not well understood. Conventional characterization techniques can confirm that complexation occurs, but they don’t provide insight into the specific binding modes or structural changes. This highlights the need for more advanced tools to study these interactions in greater detail.
This work demonstrates that Raman and infrared spectroscopy are powerful tools for studying PEI-DNA complexation. First, we use Raman spectroscopy to analyze polyamine oligomers interacting with single-stranded DNA (ssDNA). We identify a spectroscopic marker in the polyamine spectrum that is sensitive to non-covalent interactions, including electrostatic forces and hydrogen bonding. Using this marker, we show that polyamines primarily bind ssDNA through electrostatic interactions, causing DNA condensation, while also forming hydrogen bonds with DNA bases.
Next, we use infrared spectroscopy to examine the binding behavior of more physiologically relevant PEI systems, specifically high molecular weight linear and branched PEI, with double-stranded DNA (dsDNA). The IR spectra reveal that linear PEI binds DNA through electrostatic interactions that induce a conformational shift, while branched PEI primarily intercalates into DNA bases.
Together, these findings challenge the conventional view of purely electrostatic binding and provide new insight into the chemical and structural factors governing PEI-DNA complex formation. This work offers a foundation for the rational design of more effective polymer gene delivery systems.
Language
en
Number of Pages
171 p.
Recommended Citation
Mustafa, Rusul, "Developing Vibrational Spectroscopy Methods to Elucidate Polymer-Nucleic Acid Interactions for Improved Gene Delivery" (2025). Graduate College Dissertations and Theses. 2091.
https://scholarworks.uvm.edu/graddis/2091