Sarah Eaton

Topic

An examination of the use of the chemotherapy drug doxorubicin, gold nanoparticles, and radiation in combined cancer therapy

Department of Physics and Astronomy

Date & location

• Friday, August 2, 2024
• 11:00 A.M.
• Virtual Defence

Examining Committee

Supervisory Committee

• Dr. Devika Chithrani, Department of Physics and Astronomy, University of Victoria (Supervisor)
• Dr. Wayne Beckham, Department of Physics and Astronomy, UVic (Member)

External Examiner

• Dr. Mohsen Akbari, Department of Mechanical Engineering, UVic

Chair of Oral Examination

• Dr. Issa Traore, Department of Electrical and Computer Engineering, UVic

Abstract

The chemotherapy drug doxorubicin (DOX) is a widespread and effective treatment for many different types of cancer. However, it is known for causing significant and dangerous side effects due to high cardiotoxicity. Gold nanoparticles (GNPs) are a promising field of nanomedicine due to their stability, customizability, and radiosensitization properties as demonstrated using in vitro and mice models. They accumulate preferentially in tumours due to the enhanced permeability and retention effect. The combination of GNP mediated radiotherapy and DOX has the potential to deliver highly targeted and effective therapeutics while sparing surrounding healthy tissue.

This work used GNPs conjugated with PEG and RGD, DOX, and radiotherapy in combination to investigate possible synergistic cancer therapeutics. MDA-MB-231 cells were dosed for 48 hours with GNPs at a clinically relevant concentration of 7.5 μg/mL. DOX was dosed at the measured IC50 concentration of 144.4 nM for a 48 hour exposure. Radiation doses of 2 Gy and 5 Gy were used, as 2 Gy is commonly used for fractionated radiotherapy and recent clinical trials have also shown 5 Gy to be an effective fractionated radiation dose.

A cytotoxicity assay was conducted to determine the IC50 of DOX which was used as the dosing concentration for all other assays. Live cell images were taken to demonstrate the internalization of DOX and GNPs in the cells. To quantify if DOX affected the uptake of GNPs into the cells, a cellular uptake study was conducted. As previous research has indicated that DOX causes cell cycle arrest, a cell cycle assay was conducted. To assess the cytotoxicity and radiosensitization properties of GNPs and DOX, a cellular proliferation study and a clonogenic assay were conducted. Additionally, a DNA double strand break assay was conducted to assess the amount of DNA damage caused.

The cellular uptake study revealed that DOX caused an increase in GNP uptake, with (1.27±0.16)×10⁶ GNPs per cell when treated with DOX, and (0.76±0.05) ×10⁶ GNPs per cell when untreated. DOX showed evidence of radiosensitization in the proliferation assay with the combination of DOX and radiation causing a (54±2)% reduction in cell growth when 2 Gy was administered, and a (69±8)% reduction in cell growth when 5 Gy was administered. However, this effect was not synergistic. In the other assays conducted, DOX caused cell cycle arrest, extensive DNA damage, and no clonogenic growth. It was concluded that DOX was inducing senescence at the given dose.

GNPs showed some radiosensitization in the proliferation assay at 2 Gy, with (24±2)% reduction in growth after 3 days in the 2 Gy GNP sampled compared to (15±2)% reduction in growth in the 2 Gy control sample. No other significant differences in growth due to GNPs were seen in the proliferation assay. The clonogenic assay showed that 2 Gy radiation caused a (67±5)% decrease and 5 Gy caused a (97.9±0.6)% decrease in clonogenic survival of cells treated with radiation only when compared to the unirradiated control. The GNP incubated sample demonstrated some radiosensitivity in the clonogenic assay as it had a (78±3)% lower surviving fraction when irradiated with 2 Gy then the unirradiated control. The GNPs also showed toxicity in the unirradiated sample, with (30±11)% lower surviving fraction than the control in the clonogenic assay. A Bliss independence test found the GNPs and 2 Gy radiation to have independent effects. There was no significant difference between the GNP and control cells in the clonogenic assay when irradiated with 5 Gy. The DNA double strand break assay showed that 2 Gy radiation caused an increase in DNA damage foci from 2.0±0.2 to 5.1±0.5 foci per cell. No significant difference in foci was seen between the control and the GNP incubated cells.

While the results from this work did not demonstrate a conclusive benefit from the combined therapy of doxorubicin, GNPs, and radiation, the system is still of interest. Future experiments could be performed using a reduced doxorubicin concentration such as the IC20, to reduce the toxicity while still causing an effect. If a synergistic effect can be observed, it could be exploited to significantly reduce normal tissue toxicity in cancer patients while still delivering a lethal dose of chemotherapy and radiotherapy to the tumour.