**4. Rationale for Gold Nanoparticles in Chemoradiotherapy**

Radiotherapy is mainly used to control the tumor locally as discussed previously. Chemotherapy is used to control the tumor metastasis. Therefore, a combination of chemotherapy and radiotherapy (chemoradiation) is being practiced in the clinic to treat patients with locally advanced disease. Considering the variety of drugs available for cancer treatment, the possible choice of sequencing of combined chemotherapy and radiation therapy is countless, and the treatment plan differs between each patient. The standard treatment sequence refers to chemotherapy regimen before a traditional external beam radiation therapy treatment [53]. Chemotherapy used prior to irradiation is expected to cause maximal tumor regression for locally advanced tumors. The major limitation of combining chemotherapy and radiation therapy is normal tissue toxicity, since either modality can cause major normal tissue toxicity [8]. The main problems currently associated with chemotherapy are the biodistribution of pharmaceuticals, the lack of drug-specific affinity towards the tumor, limited plasma half-life, poor solubility and stability in physiological fluids, and nonspecific toxicity [126]. GNPs, due to their high surface area-to-volume ratio, as well as a large number of surface bio conjugation possibilities, are an ideal platform for delivering pharmaceutics for chemotherapy [46,127–129]. The use of GNPs as drug delivery system (DDS) can improve the pharmacokinetics, the pharmacodynamics, and the biodistribution of various drugs, as well as allow for improved targeting to reduce normal tissue toxicity. Beyond being an effective radiosensitizer, GNPs allow for a 100- to 1000-fold increase in ligand density compared to that of liposomal or polymeric DDSs [55]. Thus, the combination of the

GNPs with radiotherapy and chemotherapy is part of the natural progression of the exploration of GNPs as a complete treatment modality.

The conjugation of moieties such as chemotherapeutic agents to the surface of the GNPs can be done using various techniques. The most common method is through the use of thiol group-containing biomolecules [130]. The use of thiolated biomolecules allows for functionalization of the GNPs with various agents, such as DNA, peptides, antibodies, and proteins [131,132]. This is a very robust method, as the majority of anticancer drugs can be thiolated, so as to be compatible with GNPs as a DDS [133,134]. Furthermore, capping agents, such as carboxyl terminated PEG molecules, with a thiol bond can allow for further functionalization techniques [131,135]. A general overview of various drug loading techniques using GNPs was explored by Fratoddi et al. [136]. GNPs, due to their favorable surface chemistry, are a suitable drug carrier for use in chemotherapeutics, and may be available for use in a wide range of drug delivery applications.

GNPs have been conjugated to a large variety of cytotoxic, anticancer drugs, and combined with radiation for improved efficacy. This includes paclitaxel, methotrexate, gemcitabine, doxorubicin, docetaxel, bleomycin, and platinum-based drugs like cisplatin [133,137–141]. Many different drugs can be used for different purposes, a few of which we will expand on. For instance, the antitumor activity of cisplatin was first discovered by Rosenberg and co-workers in 1960s, when they were examining whether electrical currents affect cellular division [142]. The researchers discovered that the inhibition of cellular division observed in the study was not due to electrical current, but platinum hydrolysis products formed from platinum electrodes. They reported that *cis*-tetrachlorodiammineplatinum (IV), *cis*-[PtCl4 (NH3)2], was the potent agent responsible for the inhibition. Cisplatin is now used to treat various types of cancers (i.e., cervical cancer, non-small-cell lung cancer, ovarian cancer, germ cell tumors, osteosarcomas, etc.), with a cure rate as high as 90% in testicular cancer [143]. However, long term cisplatin usage results in drug resistance [144]. To counteract this resistance, very high systemic doses of cisplatin should be administered, which results in severe systemic toxicity and poor patient compliance, limiting its clinical use [144–146]. It was shown recently that GNPs can be used to enhance damage caused by platinum-based anticancer drugs [147,148]. Comenge et al. conjugated cisplatin to GNPs and tested the efficacy of this DDS compared to the free drug along, as shown in Figure 7A–C [147]. They found that the use of GNPs led to 300 times more platinum being encapsulated in A549 cells in vitro, and while moving to in vivo, found similar efficacy but largely absent normal tissue toxicity. Yang et al. instead used free cisplatin along with GNPs in a combined chemoradiotherapy modality in vitro, seen in Figure 7D–F [116]. An additive relationship was discovered when treated with GNPs, cisplatin, and radiation in MDA-MB-231 cells. The use of GNPs may be an important avenue to explore when integrating cisplatin into chemoradiation protocols.

Another chemotherapy agent that is limited due to high normal tissue toxicity is docetaxel (DTX). DTX is a cytotoxic member of the taxanes and is an effective antimicrotubular agent that is effective in the treatment of multiple different types of cancers including head and neck, breast, prostate, and non-small-cell lung cancer [149–152]. Docetaxel's mechanism of action is primarily through the ability to enhance microtubule assembly and stabilize free microtubules within the cytoplasm, thus preventing their depolymerization during normal cell division [153]. This has many consequences for the fate of the cell, including inhibition of progression through the cell cycle and inevitably death via mitotic catastrophe, depending on the dose [154]. Francois et al. tested DTX-conjugated GNPs on MCF7 and HCT15 cells and found a 2.5 times more efficient response compared to free DTX [139]. DTX has also been shown to block the cell cycle at the G2/M phase [155]. This is critical because the G2/M phase has shown special sensitivity to the ionizing radiation, causing more cell death [156]. Moreover, by arresting tumor cells in the M phase of the cell cycle, it synergizes the lethal effects of radiotherapy, thereby serving as an ideal radiosensitizer. Both in vitro and in vivo studies have demonstrated the synergistic effects of DTX when combined with radiotherapy [157,158]. Furthermore, it has been shown that the uptake of NPs, including GNPs, is increased when the cell population is synchronized in the G2/M phase [85,159]. This suggests the use of DTX concomitantly with other drugs or radiation,

which was tested by Bannister et al. as seen in Figure 8 [160]. DTX used as a synchronizing agent when paired with GNPs lead to higher uptake, a higher localization of the GNPs to the nucleus, and a larger, synergistic response to radiotherapy.

**Figure 7. Cisplatin and gold nanoparticles**. (**A**–**C**) Cisplatin conjugated GNPs lead to an increased deposition of platinum into A549 cells compared to the free drug in vitro, which led to less side effects in vivo with similar efficacy. Scale bar is 4 μm. (**D**–**F**) The use of free cisplatin to synergize with GNPs in a chemoradiation modality in vitro lead to a synergistic effect. Scale bar is 40 μm. Reproduced with permission from [116,147]. Copyright Public Library of Science, 2012; Copyright Multidisciplinary Digital Publishing Institute, 2018.

**Figure 8. Docetaxel and gold nanoparticles.** (**A**) Confocal imaging of GNPs (labelled in red) and the microtubule (MT) structure (labelled in green) in HeLa cells. (**B**) Confocal imaging of GNPs and MTs after treatment with 50 nM of DTX. (**C**) Radiosensitization of GNPs without DTX. (**D**) Radiosensitization of GNPs with 50 nM DTX, showing a synergistic effect. Scale bar is 25 μm. Reproduced with permission from [160]. Copyright British Journal of Radiology, 2020.

Normal tissue toxicity is an issue in escalating current dose regimes in many tumors. However, in pancreatic cancer, despite advancements in chemotherapy and radiotherapy, the current 5 year survival rate is only 9% [161]. Gemcitabine is a pyrimidine analog that is a mainstay treatment of adenocarcinoma of the pancreas, but is also used for treatment of bladder and non-small cell lung cancer [162–164]. Upon cellular encapsulation, gemcitabine is phosphorylated to its active diphosphate and triphosphate metabolites, which inhibit RR and DNA synthesis, respectively [165]. Despite its prominent use in the clinic, only a small portion of the drug is converted to its active forms. Up to 90% of the injected dose is collected from urine one week after treatment, with 75% of that being in the first 24 h [166]. The use of gemcitabine-conjugated GNPs could be an avenue for both improved uptake of the drug as well as improved efficacy in the very deadly pancreatic disease. It has been shown that 20 nm GNPs by themselves can sensitive pancreatic cell to the effects of gemcitabine by Huai et al. [167]. This is explained by showing that GNPs inhibited epithelial to mesenchymal transition and reduced cancer cell stemness—possible causes of anticancer drug resistance [168]. Furthermore, Pal et al. showed in Figure 9 that gemcitabine-conjugated GNPs that specifically target pancreatic cancer cells with a plectin-1 peptide have improved efficacy in situ compared to the free drug alone [169]. The use of gemcitabine with radiotherapy has also been shown to be more effective than the drug alone through clinical trials [170]. Thus, in the future, the addition of gemcitabine-conjugated GNPs to a radiotherapy protocol may prove highly beneficial.

**Figure 9. Gold nanoparticle mediated delivery of Gemcitabine.** (**A**) Gemcitabine-conjugated GNPs showed a significant increase in efficacy when treating mice, as measured through volume. (**B**) KI67, a marker for proliferation, also showed reduced proliferative cells when treated with the GNP complex compared to the free drug alone, signifying improved efficacy. Black = vehicle, Red = GNP, Blue = Gem and Purple = GNP-Gem. \* and \*\* denote *p* < 0.05 and *p* < 0.01 compared to vehicle-treated group respectively. Reproduced with permission from [169]. Copyright Royal Society of Chemistry, 2017.

The use of GNPs as chemotherapeutic DDSs is increasing, and the combination of chemotherapy with radiotherapy remains one of the most effective treatment modalities available in the clinic. A brief summary of recent studies, no older than 2016, involving the use of GNPs with chemotherapy and radiotherapy, can be seen in Table 1. The use of GNPs in combination with anticancer drugs and radiation is still limited in literature; however, the published work thus far shows a trend of improved dose response to the tumor coupled with reduced normal tissue toxicity. Further studies need to be completed, however, before translation to the clinic.



GNP: Gold Nanoparticle; PAC: Paclitaxel; TNF: Tumor Necrosis Factor; PEG: Polyethylene Glycol; DOX: Doxorubicin; BLM: Bleomycin; CIS: Cisplatin; GLC: Glucose; PDC: Peptide-drug-conjugate containing chlorambucil, melphalan, or bendamustine; 5-FU: 5-Fluorouracil; GSH: Glutathione; CS: Chitosan; RGD: arginyl-glycyl-aspartic acid tripeptide; DTX: Docetaxel
