Author ORCID Identifier

https://orcid.org/0009-0004-2839-2015

Date Available

8-10-2027

Year of Publication

2025

Document Type

Master's Thesis

Degree Name

Master of Science in Chemical Engineering (MSChE)

College

Engineering

Department/School/Program

Chemical and Materials Engineering

Faculty

Brittany E. Givens, PhD

Faculty

J. Zach Hilt, PhD

Abstract

Drug delivery systems (DDS) enhance drug delivery for treating gynecological cancers by increasing the bioavailability of commonly prescribed chemotherapeutics in these cancers. Gynecological cancers may originate in the ovary or fallopian tubes, uterus, cervix, and the vagina or vulva; each tissue of origin presenting unique signatures that determine the efficacy of active pharmaceutical ingredients in treating these cancers. Aside from cervical cancer, no screening mechanisms exist for gynecological cancers and therefore these cancers are typically diagnosed at later stages. Pre-clinical development for nanocarrier DDS has focused on improving existing treatments and developing DDS for novel therapies. For example, metals, metal oxides, and metal organic frameworks, have been investigated in ovarian, endometrial, and cervical cancers as alternatives to platinum- containing chemotherapeutics. Alternatively, polymeric delivery methods can improve the delivery of existing drugs by offering greater specificity for the target tissue and thus higher bioavailability. Advances in drug delivery systems enhance the therapeutic efficacy of gynecological cancer treatments, but clinical translation still requires significant stages of investigations, including in vivo studies and clinical trials.

As a potential alternative to platinum-based chemotherapy that exhibit high resistance in advanced or late-stage cancers, copper oxide nanoparticles (CuO-NPs) were investigated for their anti-neoplastic properties in vitro. CuO-NPs were selected based on their high redox potential and copper’s micronutrient properties. CuO-NPs have been known to induce reactive oxygen species (ROS) in various cell lines, both human and animal. Our study used a range of stages, grades, and metastasis of four endometrial cancer and three cervical cancer cells with CRISPR-modified knockouts of DNA repair genes to explore whether the biological characteristics impact therapeutic efficacy of these particles and compare these to a healthy epithelial control.

We explored uptake of CuO-NPs using ICP-MS, revealing an increase in relative copper content for all treatment groups. Ishikawa cells showed the greatest uptake at the 20 µg/mL treatment group and was the only cell line with functioning estrogen and progesterone receptors. In HeLa S3 cells, caveolae-mediated endocytosis and macropinocytosis were identified as uptake mechanisms of CuO-NPs, with the latter being the dominating pathway of those tested. Based on our results, each cell line exhibited unique toxicity profiles, quantified by IC50 curves and values. Further investigation revealed that viability was linked to the stage of apoptosis, with more sensitive cell lines in viability assays showing large percentages of cells in early and late apoptosis, indicating this was the dominating mechanism of cell death. Oxidative stress was investigated as a potential mechanism leading to apoptosis and revealed that all cell lines showed oxidative stress after 1 and 6 hours of exposure to CuO-NPs. However, HEK293 and KLE cells showed the highest control antioxidant potential, signifying that this may be the underlying mechanism for cell line-dependent sensitivity to CuO-NPs. Further investigation will explore pharmacological inhibitors to explore uptake differences between all cell lines. The Seahorse XF assay will be used to monitor mitochondrial respiration and glycolysis rates to determine the effect these particles have on the function of cells’ mitochondria.

Although drug delivery is essential to combating gynecological cancer, understanding the underlying causes of disease promotion in gynecological tissues through endocrine disruption can highlight the importance for strategies to prevent cancer causing mutations. The increasing environmental prevalence of micro- and nanoplastics (MNPs) spurs urgency in scientific research to understand their impact on human health. These plastic particles enter the environment through degradation of plastic products, shedding or disposal of synthetic clothing, and production runoff for consumer or industrial use. Nanoparticles and nanoplastics act as endocrine-disrupting chemicals (EDCs) by interfering with hormone signaling, which can cause an abundance of adverse health outcomes including hormone-linked cancers. This study investigated the impact of polystyrene nanoparticles (PS-NPs) on the proliferation of HeLa S3 cervical cancer cells, including two CRISPR-modified knockouts for DNA repair pathways: MLH1 and MSH2.

PS-NPs and fluorescent PS-NPs (FPS-NPs) were characterized to determine size, morphology, and surface charge, which all influence cellular uptake and intracellular trafficking. At low concentrations, confocal microscopy revealed particles were localized to the cell surface and cytoskeleton; quantitative uptake from lysed cells followed a dose- dependent response. Two methods of cytotoxicity were investigated: mitochondrial and esterase activity. The results indicated that there was minimal effect in cell viability using both viability methods, which followed a dose-dependent response. Likewise, results from apoptosis assays revealed a small increase in the percentage of cells in the apoptotic regions for both the HeLa S3 parent and MLH1 knockout cells treated with mono- and bi-layers of PS-NPs. However, measurement of oxidized and reduced glutathione revealed no change to oxidative stress levels after 24 hours of exposure. Further investigation on how EDCs interfere with hormone signaling will provide crucial insights into how MNPs impact gynecological cancers and healthy tissues. Future studies will include investigation into gene expression differences using qPCR and additional MNP models, such as mechanically fragmented polyethylene terephthalate (PET) particles as potential EDCs.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.306

Funding Information

This research was supported by Oak Ridge Associated Universities in 2022-2024, University of Kentucky Pigman College of Engineering Fellowship in 2023-2024, and University of Kentucky Center for Appalachian Research in Environmental Sciences (P30 ES026529) in 2024-2025.

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Available for download on Tuesday, August 10, 2027

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