**3. Gold Nanoparticles as Radiosensitizers**

The use of high atomic number (Z) material to enhance radiation dose has been studied for more than 50 years. The interest in using high-Z material stems from the production of secondary electrons, such as photoelectrons, Auger electrons, and Compton electrons. These secondary products are effective at damaging DNA as well as ionizing surrounding water molecules, forming free radicals [110]. While the atomic number of tissue is approximately Z~7.5, materials with a higher atomic number used in the past such as Iodine (Z = 53) and gold (Z = 79) have a larger cross-section for absorption of radiation. For example, it was demonstrated in vitro that incorporating iodine into cellular DNA using iododeoxyuridine enhanced radiosensitivity at keV ranges by a factor of three [111]. The outcome of the in vitro study was also seen in an in vivo study, where an intratumoral injection of iodine and 200 kVp X-ray radiation suppressed the tumor growth by 80% [112]. In addition to having a great difference in mass attenuation between gold and soft tissue, gold has been shown to be biocompatible, simple, and economical to manufacture in many different shapes and sizes [113].

Radiation dose enhancement due to GNPs was first demonstrated using 1.9 nm GNPs in a mouse model, in one of the pioneering studies in GNP-mediated radiation dose enhancement by Hainfeld et al. [29]. A radiation dose of 30 Gy with 250 kVp X-rays to subcutaneous tumors in mice resulted in a significant decrease in tumor volume. However, the concentration of gold in this study was considerably high, at 2.7 g Au/kg body weight, which is not clinically feasible. Furthermore, the use of kV energies, while allowing for prominent photoelectric absorption in gold, is hindered due to the reduced penetration for deep-set tumors. Thus, as previously discussed, optimization of the internalization of the GNPs into the tumor cells, both in-vitro and in-vivo, is required for ideal efficacy. Whenever gold was internalized in vitro, radiosensitization was achievable at MV energy ranges, at concentrations as low as 1 ng/g [25,114–116]. This was demonstrated by Chithrani et al. in Figure 5A–C, which found a 17% increase in radiosensitization at 6 MV with 50 nm spherical GNPs [25]. When moving to an in vivo environment, radiosensitization was seen at a delivered dose of 10 μg/g of body weight [117]. This was accomplished by Wolfe et al. using targeted GNRs, as seen in Figure 5D–F, where there was a 36% increase in radiosensitization in vitro in PC3 cells, and a significantly enhanced tumor-growth delay when treated in vivo [117]. The treated dose is a ~1 <sup>×</sup> 106 improvement over the original treatment seen in Hainfeld's pioneering study. The addition of targeting and improvements in the optimization of uptake has allowed significant progress in facilitating the progress of gold nanomaterials to the clinic.

**Figure 5. Radiosensitization due to gold nanoparticles.**(**A**–**C**) Spheroidal GNPs improve radiosensitization in vitro, with the largest effect occurring with 50 nm GNPs, as they have the optimum uptake. This can be seen both through clonogenic assays as well as through imaging of double strand break foci with confocal microscopy. (**D**–**F**) GNRs displayed increased radiosensitization when targeted towards prostate cancer cell lines both in vitro through a clonogenic assay as well as in vivo through tumor volume measurements in a mouse model. Reproduced with permission from [117]. Copyright Elsevier, 2015.

GNPs localized intracellularly increases the probability of ionization events leading to local enhanced deposition of energy causing more damage to tumor cells [25]. The physical mechanism of GNP radiosensitization, seen in Figure 6A, occurs within the first nanoseconds of exposure, and is based on the difference in mass energy absorption coefficients between gold and soft tissue, enabling dose enhancement. The range of electrons released from GNPs is short, only a few micrometers, causing highly localized ionizing events. Thus, to achieve any enhancement from GNPs in radiation therapy, GNPs must be delivered and internalized specifically by tumor cells.

**Figure 6. Radiosensitization and radiobiological e**ff**ects.** (**A**) Schematic showing chemical mechanism of GNP radiosensitization. While the radiation causes direct and indirect damage (yellow and red stars, respectively), there can be induction of secondary electrons and reactive oxygen species through gold nanoparticles. This can lead to damage to the DNA as well as secondary parts of the cell, such as the mitochondria. (**B**) GNPs can influence the cell through generation of reactive oxygen species, DNA damage, as well as cell cycle and bystander effects. Reproduced with permission from [110,118]. Copyright Springer Nature, 2016, 2017.

The chemical mechanism of GNP radiosensitization occurs through the radiochemical sensitization of DNA by increasing catalytic surface activity and increasing radical generation from the GNP surface [60]. Despite the prevailing notion that GNPs are chemically inert, there is a growing body of evidence that, due to the electronically active surface of GNPs, they are capable of catalyzing chemical reactions [119]. Catalysis by GNPs occurs mainly through surface interaction with molecular oxygen, generating free radicals [60]. This seems more evident in small GNPs (<5 nm in diameter) where surface to volume ratio is greater [120]. When combined with irradiation, the catalytic effects appear to be enhanced, with smaller GNPs with larger surface areas yielding more ROS [121]. However, it has been shown that at all energy levels, the dose enhancement observed cannot be simply explained by physical or chemical mechanisms [122]. To explain this, a radiobiological effect must be occurring.

The main radiobiological mechanisms involved in the cell's response to irradiated GNPs results are the production of reactive oxygen species (ROS), oxidative stress, DNA damage induction, potential bystander effects, and cell cycle effects, as explained by Rosa et al. in Figure 6B [118]. Oxidative stress can cause cellular damage to the cell, including the oxidation of lipids, proteins, and DNA, which can result in apoptotic and necrotic cell death [123]. The mitochondria do appear to play a role, and the data indicate loss of function due to high intracellular ROS levels. It has been shown that the use of 1.4-nm triphenyl monosulfonate (TPPMS)-coated GNPs resulted in a loss of mitochondrial potential through elevated oxidative stress, resulting in necrotic cell death [122]. There have also been studies suggesting that GNPs may cause cell cycle disruptions and induce apoptosis. Radiosensitivity varies throughout the cell cycle with S phase being where a cell is most radioresistant and G2/M phase being most sensitive [124]. This could also depend on cell type, expression of cyclin kinases, and NP characteristics such as coating and size. For example, the use of 1.9 nm GNPs in DU-145 and MDA-MB-231 resulted in an increase in sub-G1 population in DU-145 population but not in MDA-MB-231 [125].

The biocompatibility of GNPs has already been tested in a phase I clinical trial. Furthermore, both in vitro and in vivo studies have shown the possibility of using GNPs as a radiosensitizer at clinically feasible concentrations, as discussed previously. Radiotherapy can also be combined with chemotherapy (chemoradiation) in cases where the tumor is not localized anymore, but metastasized as well, or to reduce potential micro-metastases spread. We will discuss the recent research conducted towards adding GNPs to this chemoradiation protocol in the next section.
