Gold nanoparticle uptake in synchronized cell populations and the effect on radiation sensitization

Abstract

To overcome the challenge in radiation therapy of delivering the prescribed dose to cancer cells while sparing normal tissue, preferential introduction of high Z material to tumour cells works as a method of radiation sensitization. Gold nanoparticles (GNPs) are very useful in this respect. It has been shown that the size, shape, and surface properties of GNPs affect their cellular uptake. Manipulation of the cell cycle to arrest cells at different stages offers a unique strategy to study the molecular and structural events as the cell cycle progresses. To optimize delivery of GNPs into tumour cells and enhance the effect of radiosensitization, nanoparticle (NP) uptake in synchronized populations of MDA-MB-231 breast cancer cells was investigated.

Populations of MDA-MB-231 cells were first synchronized in S-phase using double-thymidine block, and allowed to progress through cell cycle in synchronization. Synchronized cells were incubated with 5 nm GNPs, 15 nm GNPs, 46 nm GNPs and two formulations of lipid NP encapsulated 5 nm GNPs. Uptake of NPs was visualized using hyperspectral optical imaging and quantified with inductively coupled plasma mass spectrometry (ICP-MS). Following internalization of GNPs, cells were irradiated with 6 MV photon beams from a linear accelerator, and the survival fraction and induced deoxyribonucleic acid (DNA) damage were studied.

Cell cycle analysis after a double-thymidine block showed that the cell population was well synchronized. Uptake of NPs was 1.5-2 times higher in synchronized cell population compared to the control where cells were at different stages of the cell cycle. Clonogenic studies were used to evaluate the cell survival following radiation treatment. After a dose of 2 Gy, there was a decrease in cell survival fraction in synchronized cells treated with GNPs prior to radiation treatment compared to unsynchronized cells (control) indicating GNP-mediated dose-enhancement. The protein γ-H2AX, which is recruited to sites of DNA double strand breaks, was fluorescently labeled to evaluate damage due to the radiation treatment. Our results show more DNA double strand breaks in cells treated with GNPs prior to radiation. Interaction of ionization radiation with GNPs inside of cells produce secondary electrons. These secondary electrons can interact with water molecules and produce additional free radicals. These low energy electrons and free radicals interact with important cell structures and could cause cellular damage. Cell cycle synchronization has been shown to enhance GNP/PEG/RGD uptake in MDA-MB-231 cells resulting in greater cell radiosensitization and cellular damage. Cell synchronization is therefore an additional method available that can be employed to improve GNP uptake in cells.