Delivering foreign molecules into living cells is a broad and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via non-viral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. In this thesis, an array of solid silicon lances was tested by nanoinjecting tens of thousands of HeLa cancer cells simultaneously. Several molecule types were injected in different tests to understand cell uptake efficiency and cell viability. Voltage was used to determine the impact of an electric field on molecule delivery. Propidium iodide, a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers propidium iodide into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78%-91% viability. Using similar protocol as in propidium iodide experiments, plasmid DNA containing the code for a fluorescent protein was nanoinjected into HeLa cells, resulting in an average expression rate of up to 0.21%. Since gene expression only occurs in cells which have integrated DNA into the genome in the nucleus, a different DNA detection method was developed to determine total DNA count in cells following nanoinjection. DNA strands tagged with a radioactive isotope were nanoinjected into HeLa cells. Liquid scintillation was employed to quantify and discriminate between DNA delivered to cells and DNA that remained in solution around cells following nanoinjection. The largest average amount of DNA delivered to cells was 20.0 x 10^3 DNA molecules per cell. Further development of the radioactive nanoinjection process is needed to more fully understand the parameters that affect DNA delivery efficiency. In all experiments with propidium iodide and DNA molecules, low accumulation voltage, coupled with a short pulsed release voltage, resulted in the greatest molecule delivery efficiencies when compared to tests without voltage or with a constant voltage only. Lastly, an automated nanoinjection system was developed to eliminate variability in user applied nanoinjection force. The automated system was found to reduce variability in average propidium iodide uptake values by 56%. In conclusion, experimental testing of the multi-cell nanoinjection process has shown promising molecule delivery results into human cells, suggesting that further optimization of the process would have positive implications in the field of academic and clinical gene therapy.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-5034 |
| Date | 05 May 2014 |
| Creators | Lindstrom, Zachary Kendall |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
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