Date of Award

8-28-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Applied Science

First Advisor

Alexandru Biris

Abstract

Since the inception of nanoparticles, they have affected almost each and every field of modern science and technology both in terms of research and application. Due to its subcellular level size and ease of modification for biological and medical purposes, nanoparticles have contributed greatly in various field of biomedical reaserch including cancer research. In this dissertation, emphasis has been given on an important area of research of a multi-modal anticancer therapeutic approach using carbon-based and magnetic inorganic nanoparticles. Cancer is a complex disease that has been known to be generated by mutations at the genetic level, and continue to accumulate multiple mutations in order to survive human body's natural protective pathways. Among all the mutations that occur in different types of cancer, p53 gene mutation is one of the most important and common one. As this (p53) protein is responsible for programmed cell death process, most forms of cancer cells show abnormal behavior of p53 protein. Ethylenediamine functionalized single wall carbon nanotubes (SWNTs) have been used to deliver a functional copy of p53 gene in a plasmid construct, to human breast cancer cell line MCF-7, in order to restore the activity of p53 protein, which in this case is extremely short-lived. The attachment of the plasmid on the SWNTs was determined by atomic force microscopy. The nanutobe has successfully delivered the plasmid into the MCF-7 cell which follows the expression of the p53 protein into the cell as evidenced by the expression of Green fluorescence protein which was tagged to p53 plasmid. Upon expression, the functional activity of the p53 protein was found to be significantly restored as after 72 hours of incubation ~40% of cancer cells were apoptotic. Apoptosis was further determined by caspase assay. The major problem of conventional chemotherapy is the toxicity that it causes to the normal healthy tissues of the body resulting in deadly side effects. In chapter 3, we have used SWNTs to accomplish the targeted delivery by functionalizing it with human epidermal growth factor (EGF). As EGF receptor is over expressed in many of the cancer cells, it is possible to deliver any chemotherapeutic agents selectively to those cancer cells. We used EGF conjugated to SWNTs for targeted delivery to PANC-1 cells. Results indicate EGF-functionalized SWNTs accumulate more into PANC-1 cells compared to only SWNTs only. Upon targeting, Raman spectroscopy and ELIZA assay were used to determine the association and dissociation pattern of the targeted SWCNTs. 2D-Raman mapping was used to show the higher accumulation EGF targeted SWCNTs to PANC-1 cells. Later on NIR laser (808 nm) was used to heat up the SWNTs, and the heat was able to kill almost 90% cancer cells, which is 40% more compared to untargeted SWNTs. In chapter 4, we have explored the multimodality of nanotechnology by using EGF-functionalized iron cored, carbon shelled (FeC) magnetic nanoprticle that can generate heat when exposed to radiofrequency (RF). This process called hyperthermia, which is an effective way to kill different cancer cells as cancer cells are most susceptible to heat increase beyond a threshold level compared to normal cells. By targeting the nanoparticles this process was made more efficient by selectively populating magnetic nanoparticles specifically in cancer cells only. Two different cells line, MCF-7 and PANC-1 were incubated with the magnetic nanopartilces for 24 hours followed by exposure to the RF (350KHz, 5kW) for 10 min. EB/AO and flow-cytometry were used to determine the apoptotic and necrotic cells. EGF targeted FeC was superior in apoptosis induction in both cell lines, while the untargeted nanoparticles showed little effect. The MCF-7 cells were more vulnerable to the targeted FeC nanoparticles compared to PANC-1 cells. Caspase-8 and caspase-3 assays were done to provide the involvement of these two capsizes in two different cell lines. As cancer is a very complex disease often multiple therapeutic approaches when applied together work more efficiently rather than a single one. To enhance the therapeutic efficiency of anticancer drug, two FDA approved anticancer drugs doxorubicin and erlotinib were loaded on FeC nanoparticles (Chapter 5). The loading capacity was determined by UV-Vis spectroscopy. MTT assay was also used to determine the loaded and unloaded forms of drug. RF treatment was used to generate hyperthermia and which also helps in releasing the drug from nanoparticles. Flow-cytomertic detection of apoptosis showed above 90% cells died after combined hyperthermia and doxorubicin treatment. Graphene is a 2D carbon nanoparticle that has been proven to be an excellent carrier of chemotherapeutic drugs. In chapter 6, -COOH functionalized graphene (FG) was used to deliver a potent but extremely hydrophobic and water insoluble drug, parthenolide (PTL), to PANC-1 cells. WST-1 assay showed significant lowering of the IC50 value when PTL was delivered with FG compared to PLT alone. Results also indicate, Graphene mediated delivery of PTL increased intracellular reactive oxygen species (ROS) leading to disruption of mitochondrial membrane potential (MMP) and finally apoptosis. JC-1 assay was used to determine the MMP disruption. Flow-cytometry and caspase assay and fluorescence measurement were done to verify the apoptotic process.

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