Honors Program


Date of this Version


Document Type



Seefeld, Madison. Improving DNA Loading and Transfection Outcomes of Bacterial Outer Membrane Vesicles Using Calcium Chloride. Undergraduate Honors Thesis. University of Nebraska-Lincoln. 2022.


Copyright Madison Seefeld 2022.


Gene therapy is a promising therapeutic strategy to combat genetic and infectious diseases. Viral vectors provide the highest transfection efficiency but suffer from high toxicity and immunogenicity. Nonviral vectors induce lower immunogenicity and toxicity but lack transfection efficiency. Therefore, new nonviral vectors are needed. There are many administration routes available, but this study utilizes the oral route due to high patient compliance, ease of dosing, and no need for trained medical personnel. While the oral route has many benefits, it also poses a new set of barriers. In this work, a novel oral gene delivery system utilizing bacterial outer membrane vesicles (OMVs) loaded with pDNA via electroporation was tested. OMV-DNA nanocarriers (OMV-DNA NCs) were produced by mixing OMVs and pDNA in phosphate-buffered saline (PBS) in a 1:1 µg pDNA to µg OMV protein ratio, calcium chloride was then added to reach final concentrations of 1 mM, 25 mM, or 50 mM. The inclusion of calcium chloride was based off of previous work that has demonstrated calcium chloride increases binding of DNA to LPS, a component of the membrane of OMVS, and calcium ions increases nonviral transfection levels when added to cell culture media. This delivery system resulted in higher pDNA loading efficiency and significantly higher transfection levels when compared to control OMV-DNA NCs produced without CaCl2. Additionally, the OMV-DNA NCs did not induce higher levels of toxicity, but rather significantly increased cell viability when compared to un-transfected control cells. The work of this thesis demonstrates the potential for our OMV-DNA NCs to be used as a novel oral gene delivery system.