This project explored a gene-regulated chemotherapy using a silver nanoparticle (SNP) conjugated with deoxyribozyme (DNAzyme) oligonucleotides which target a mutated gene in select cancer cells, sensitize them to doxorubicin treatment. Light exposure to the SNP-DNAzyme conjugates disengages the oligonucleotides and permits specific cleavage of the Kirsten Rat Sarcomal Oncogene Homolog (K-RAS) mRNA. These conjugates could provide spatiotemporal specificity in killing only those photoexposed cells with the mutant gene. Synthesis, functionalization and characterization of citrate and hydroxypropyl cellulose SNP conjugates confirmed attachment and photolytic release of the thiol-modified 10-23 DNAzyme. Gel electrophoresis was used to demonstrate DNAzyme photoactivation, showing greater K-RAS RNA degradation when disengaged compared to the SNP-tethered form. DNAzyme in the tethered form was also protected from DNAse degradation compared to photolyzed DNAzyme. Characterization of the toxicity and localization of the nanoparticle drug delivery system constructed for the release of a photolabile DNA oligonucleotide was checked within several sets of cells to check for temporal and spatial control efficiency. MTS, alamar blue, and flow cytometry assays were performed to assess cell viability in several sets of cell cultures, including HEK293 and MCF-7 (wildtype K-RAS), SW480 and MDA-MB-231 (mutant K-RAS), and 3T3 (negative control) lines. Following the 5-day experimental protocol involving staggered treatment with SNP-DNAzyme, UV light, and doxorubicin, no cell group showed the intended pattern of necrosis in mutant K-RAS cells without morbidity in controls or partial treatments. Thus further evaluation of K-RAS+/- cells which respond consistently in viability assays is necessary before this strategy can be deemed of potential as a targeted therapeutic.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-11152014-131055 |
| Date | 25 November 2014 |
| Creators | Moll, Alyson Nanette |
| Contributors | Monroe, Todd, Hayes, Daniel, Sabliov, Cristina, Liu, Zhijun |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-11152014-131055/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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