Application of nano‐radiosensitizers in combination cancer therapy

Abstract Radiosensitizers are compounds or nanostructures, which can improve the efficiency of ionizing radiation to kill cells. Radiosensitization increases the susceptibility of cancer cells to radiation‐induced killing, while simultaneously reducing the potentially damaging effect on the cellular structure and function of the surrounding healthy tissues. Therefore, radiosensitizers are therapeutic agents used to boost the effectiveness of radiation treatment. The complexity and heterogeneity of cancer, and the multifactorial nature of its pathophysiology has led to many approaches to treatment. The effectiveness of each approach has been proven to some extent, but no definitive treatment to eradicate cancer has been discovered. The current review discusses a broad range of nano‐radiosensitizers, summarizing possible combinations of radiosensitizing NPs with several other types of cancer therapy options, focusing on the benefits and drawbacks, challenges, and future prospects.


| INTRODUCTION
The last decade has seen many different kinds of nanoparticles (NPs) undergoing investigation for various types of cancer treatment, including drug delivery, gene therapy, photodynamic therapy, photothermal therapy, etc. 1 Due to the size of the NPs (1-100 nm), they have a large surface area-to-volume ratio, which allows them to absorb substantial amounts of drugs and quickly disperse throughout the bloodstream. Their larger area endows them with unique features, and improves their mechanical, magnetic, optical, and catalytic properties and thus increases their broader medicinal use. 2 The heterogeneity of cancer and the multifactorial nature of its pathophysiology has led to the investigation of many different treatment approaches. Although each of these approaches has shown some promising results both in the laboratory and in the clinic, no definitive treatment to eliminate cancer has yet been established. 3 Therefore, researchers have attempted to combine two or more different methods into a single integrated approach to increase the success of treatment. 3 Combination therapy is a treatment strategy that combines two or more therapeutic agents and is the cornerstone of today's cancer treatment. 4 Combining various therapies to target cancer increases the effectiveness compared with each therapy used alone Therefore, it is crucial to find more effective methods for integrated therapy that are also economically viable. 4,5 Conventional cancer treatments non-selectively target all actively proliferating cells, leading to the destruction of both healthy and cancerous cells and consequent toxicity.
For some types of cancer, the best treatment is a combination of surgery, radiation, and chemotherapy, and possible other drugs. Surgery or radiation therapy treats locally confined tumors, while chemotherapy drugs also kill cancer cells that have spread to distant sites. In some cases radiation therapy or chemotherapy is given before surgery (neoadjuvant) to shrink the tumor, thus improving the likelihood of complete surgical removal of the tumor. 4 Radiation or chemotherapy after surgery (adjunctive therapy) is designed to destroy any remaining cancer cells. 6 The stage and type of cancer govern the choice of the optimum type of treatment. 4 Radiation therapy combined with either (or both) surgery or chemotherapy is the mainstay of cancer treatment. This involves the transfer of high intensity and accurate beams of ionizing radiation to tumor tissue, resulting in the death of tumor cells. The heterogeneous structure of a bulky tumor requires a high radiation dosage, which causes damage to healthy tissues. 7 The term radiosensitizer refers to any agent that can increase the efficiency and effectiveness of radiotherapy. During this process, the existing limitations of radiotherapy are identified and targeted to address them. The present review covers a wide range of radiosensitizing agents and discusses the state-of-the-art and future prospects. Many combination therapy strategies are discussed, including the advantages and disadvantages, challenges and future perspectives.

| RADIOSENSITIZING AGENTS
The mechanism of radiotherapy is classified into two types: direct and indirect effects. Direct damage is caused by the interaction of ionizing radiation without any intermediaries for disrupting biomolecule structure. This mainly affects single-stranded and double-stranded DNA molecules. The indirect effects are caused by the ionization of water in human tissue to produce hydroxyl radicals and other reactive oxygen species (ROS) that can also damage biomolecules and DNA. 8 Radiosensitization is a process that increases the sensitivity of cancer cells to damage caused by radiation exposure. At the same time, it reduces the potentially harmful effects on the molecular and cellular structures of the surrounding healthy tissue. 9 Hence radiosensitizers are exogenous agents that increase the effects of radiation therapy. Over the past few years, there has been a significant interest in using advanced formulations to enhance the effects of radiotherapy, especially the use of metal-based nanoparticles. 7,8,10 Radiosensitizer agents can be subdivided into three categories based on their structure: small molecules; macromolecules; nanomaterials. In addition to small molecule and nano-radiosensitizers, macromolecules such as miRNAs, proteins, peptides, and oligonucleotides are also able to increase radiation sensitivity. 9

| Small molecule radiosensitizers
Small molecules were studied at the very beginning of radiosensitizer discovery research. 11 Subsequently, some small molecules were discovered that had promising results from the beginning and are now being used clinically ( Figure 1). 5,12 Since then, a deep understanding of the molecular mechanisms of radiation therapy, and the signaling pathways associated with radiation sensitivity, have led to the production of drugs that act as radiosensitizers. Some of these can act on other pathways such as hypoxia-response and cytokines. 12,13 Other types of chemical radiosensitizers, such as pseudo-substrate, molecules that affect cell signaling, targeted transduction systems, and molecules that suppress radioprotective and repair properties, have also been developed, and some are in clinical trials. 13 Cells in the process of division and undergoing DNA synthesis are unable to differentiate between thymine and its halogenated analogues, hence the newly synthesized DNA can act as a "pseudo-substrate" ultimately leading to cell death.
Numerous signaling pathways related to apoptosis, metastasis, DNA repair, protein degradation, and other processes, can affect the effectiveness of radiotherapy. Small molecules that regulate vital pathways, such as DNA repair inhibitors and cell apoptosis activators, can enhance the efficiency of radiotherapy. Some compounds, such as the bioreductive drug Rsu1069, have dual or multiple effects, and can therefore sensitize cells not only to oxygen, but also disrupt signaling pathways and prevent DNA repair. With the advances in understanding of the mechanisms of radiation resistance, it became clear that multiple signaling pathways are associated with radiation sensitivity, providing more targets to improve the effectiveness of radiotherapy. 12 Studies on radiosensitivity-related pathways can provide new targets for radiosensitization protocols. 14 Small molecule drugs are easily modified and have well-understood evaluation systems for preclinical and clinical trials, which helps their rapid evaluation. Pharmacokinetics and pharmacodynamics are used in the design and screening of smallmolecule radiosensitizers to improve drug activity. The introduction of new methods such as computer-aided design (CAD) and virtual screening has accelerated the development of radiosensitizers. In addition, emerging nanostructures and macromolecules, which act as radiosensitizers, have shown some promising results. 12,14 2.1.1 | Oxygen and oxygen-mimetics It is known that tumor cells located in a hypoxic microenvironment are more resistant to radiation than those in a normal oxygen microenvironment. The occurrence of hypoxia in the tumor microenvironment is one of the significant limitations of radiotherapy. 15 The oxygen enhancement ratio (OER) or oxygen enhancement effect refers to increasing the therapeutic or destructive effect of ionizing radiation due to the presence of oxygen. This is called the oxygen effect, especially noticeable when cells are exposed to a dose of ionizing radiation. 12,16 Oxygen is a powerful radiosensitizer that enhances the formation of ROS and free radicals due to its electronic structure. After irradiation of an oxygenated tumor, energy transfer leads to radiolysis of water, with the initial formation of a radical ion, which after reacting with another water molecule, forms highly reactive hydroxyl radicals. Oxygen reacts with hydroxyl radicals to form peroxides, and then peroxides can cause permanent damage to cells and DNA. 9,12 The hypoxic conditions in the tumor microenvironment increase the resistance of cancer cells to damage by ROS and free radicals, and by altering signaling pathways to increase radiation resistance ( Figure 2). For example, cells undergo apoptosis via the p53 pathway under normal conditions, while under hypoxic conditions, other interconnected pathways including HIF-1α, VEGF, glucose transport, and glycolysis are activated. 7,12 Oxygen mimetics can imitate the chemical properties of molecular oxygen, yet they are designed to have higher electron affinity and better diffusion than oxygen. These oxygen mimetics can be introduced into the tumor environment, causing DNA damage and increasing cancer cell death during radiation therapy. Oxygen mimetics, also called true radiosensitizers, are of various types, and the most common types are nitrogen-containing compounds, such as nitric oxide, nitrobenzene, nitroimidazole, etc. 7

| Hypoxia-specific compounds
Although oxygen and oxygen-mimetics have been investigated for more than a decade, and significant progress has been made in these fields, they still face some obstacles and challenges. 17 These compounds often produce free radicals under the influence of radiosensitizing incorporating oxygen atoms either in oxygen molecules or other nitro groups. Lack of tumor specificity, lack of different structure types, and side effects of using hyperbaric oxygen or active nitro groups necessitate further improvement and finding other kinds of radiosensitizers.
Due to the hypoxic conditions existing inside the tumor, agents that exert preferential toxicity under hypoxic conditions could be used as radiosensitizers. Some aromatic and aliphatic N-oxides, quinones, transition metal complexes, and nitro-compounds, have bio-reductive properties, showed promising synergistic effects when combined with radiotherapy (RT). 18 The most prominent agent in this group is tirapazamine (TPZ), which when reduced to its more active metabolites, induces double strand breaks (DSB), single strand breaks (SSB), and damage to nucleic acid bases in DNA of tumor cells under hypoxic conditions. Similarly, SN30000 a biosimilar analog of TPZ, can exert cytotoxicity after being metabolized by hypoxia-activated reductases. [18][19][20][21] AQ4 (anthracenedione) is another agent with a high affinity for DNA which has shown promising results in preclinical and clinical trials. 22 Its pro-drug AQ4N, undergoes hypoxia-sensitive reduction allowing it to act as a radiosensitizer in the hypoxic tumor environment. [23][24][25] A class of nitro-compound which are used as radiosensitizers is nitroimidazoles, particularly RSU1069 and its prodrug RB6145, with high electron affinity and ability to be reduced. 26,27 RSU1069 is a well-known radiosensitizer which acts through electron reduction in hypoxic cells. 28 RB6145 was developed after RSU1069 was found to show gastrointestinal toxicity, and has similar therapeutic effects but is more tolerable. 29 The radiosensitizing effect of both drugs are improved when combined with photodynamic therapy or hyperthermia. 29

| Pseudo substrates
The incorporation of compounds similar to nucleic acid bases into new DNA strands, leads to disruption in several vital processes of cells, especially DNA replication, and has been called pseudo substrates. The most prominent of these are halogenated analogs of nucleotides, such as bromodeoxyuridine (BrUdR) or iododeoxyuridine F I G U R E 2 Tumor microenvironment and cellular pathways affected by hypoxia. (a) Oxygen concentration as a function of depth in tumor and (b) Sub-cellular effect of oxygen concentration on cancer cells treated with radiation therapy.
(IUdR). The proliferating cells in the tumor cannot distinguish between these compounds and natural thymidine, therefore they are incorporated into newly synthesized DNA molecules. Studies have shown a correlation between incorporation of BrUdR, number of DNA strand breaks, and clonogenic survival in the context of RT. 30 34 and BKM120 and BEZ235, which target PI3K pathways and increase the sensitivity of tumor tissue to irradiation. 46,47 However, some of these drugs have more than one effect through different pathways, such as RSU1069, which inhibits DNA repair, along with the aforementioned hypoxia-activated effects.

| Compounds suppressing radioprotective pathways
Mammalian cells contain a variety of reducing agents, which act to repair the damage induced by naturally produced free radicals. Glutathione (GSH) contains thiol groups and acts as an electron-donating molecule, neutralizing free radicals inside cells. 48,49 Depletion of thiol groups could be another strategy for sensitizing tumors to RT. Binding to intracellular thiol groups using MnTE-2-PyP, or inhibition of thiol production by L-S-buthionine sulfoximine lowersthe GSH content of cells and promotes the effects of RT. [50][51][52] Inhibition of other oxidoreductases, including superoxide dismutase (SOD), glutathione reductase, and thioredoxin reductase, could also impede DNA repair ability, and promote RT efficacy. 53-55

| Macromolecules
Macromolecules including, microRNAs (miRNAs), small interfering RNAs (siRNAs), oligonucleotides, peptides, and proteins can all be used to alter the radiosensitivity of cells (Figure 3). Oligonucleotides can bind to DNA through complementary binding, while siRNAs and miRNAs can lead to gene silencing, and reduce the expression of DNA protective molecules or apoptosis inhibitors. 12

| Oligonucleotides and siRNAs
Currently, siRNAs and antisense-oligonucleotides, up to 25 nucleotides in length, can be rationally designed and efficiently synthesized.
siRNAs are double-stranded RNA molecules that can interfere with and degrade mRNAs after transcription. 76,77 siRNAs can be used for silencing the genes that contribute to radioresistance. An antisense oligonucleotide against telomerase reverse transcriptase is an example of the radiosensitizing function of oligonucleotides. 78 Knockdown of survivin by siRNA negatively modulated the inhibition of caspase activation, leading to increased apoptosis after RT. 79 Similarly, siRNA-mediated knockdown of HuR mRNA resulted in enhanced radiosensitivity. 80

| Protein and peptides
Many proteins and peptides have been used in cancer treatment, especially in the form of monoclonal antibodies or small peptides with affinity to tumor-associated antigens. The anti-cancer effects are produced by the inhibition of specific pathways, which can also affect the radiosensitivity of cells. For instance, SYM004 (epidermal growth factor receptor targeting antibody) could inhibit DSB repair and increase radiosensitivity through downregulation of the MAPK pathway. 41 Similarly, the monoclonal antibody AIIB2 showed promising effects on head and neck squamous cell carcinoma by inhibiting DSB repair following inhibition of integrin β1. 81 Another major application of proteins and peptides is the targeted delivery of drugs or radionuclides. Peptides have been used to precisely deliver radionuclides to tumors as a form of brachytherapy. 82,83 Radionuclides can be incorporated in a peptide receptor strategy. 84,85 Combining peptides for highly accurate drug delivery along with nanomaterials for a high drug loading capacity synergistically increases the effectiveness of the radiosensitizing agents. 86

| nanomaterial radiosensitizers
In recent years, utilizing various nanomaterial formulations (especially metal NPs) to enhance the tumor's radiation dose has increased significantly. Several nanomaterials such as metal NPs, 94 quantum dots, 95 superparamagnetic iron oxides, 96 and non-metallic NPs 97 have been used to improve tumor radiation dose due to their unique physical and chemical properties. The use of nanomaterial radiation sensitizers is known as NP enhanced X-ray therapy. These NPs are an excellent tool for cancer diagnosis, 98 imaging, 99 and treatment. 100 Dense metal particles selectively scatter and absorb high-energy rays such as gamma/X-rays to better target the cellular components of tumor tissues. Although metal films and microparticles do not diffuse well in tumor tissue, NPs provide more cross-sectional area to interact with radiation photons. Employing NPs reduces the dose of radiation therapy, and as a result, reduces damage to healthy tissue. 100 Schematically representative of clinical trials on these nanomaterials following intravenous or intratumoral injection and subsequent cellular effect is shown ( Figure 4).
Amongst multiple nanomaterials, integrating high-z materials (such as gold, hafnium, bismuth, etc.) in cells causes a higher efficiency for cell damage caused by radiation. These NPs have chemical stability, slow metabolism, selective sensitivity, and significant effect at low doses. 101,102 For example, the cells grown on the gold film have increased the radiation dose several times. In addition, the injection of gold microparticles in tumor tissues caused a significant decrease in  The radiosensitizers based on drugs and macromolecules utilize the biological routes for sensitizing the cancer cells toward radiation. However, they are not able to profit from physical interaction of the high energy photon and any chemical reactions. High-Z NPs could be designed and delivered to target cells or tumors to act as radiosensitizers, due to their high radiation absorption cross-section compared with the surrounding soft tissue, increase the radiation dose received by the tumors. 7,100,104 While the biological effects of the high-Z nano-radiosensitizers are limited (vs. physical enhancement), simultaneous administration of drug or macromolecular radiosensitizer will increase the treatment efficiency. Of note, utilizing pharmaceutical approaches for loading and active/passive release of the molecular and macromolecular radiosensitizers in nanoplatforms will enhance their efficiency. In section 3, high-z nanoparticle will be discussed in detail.

| HIGH-Z NP-BASED RADIOSENSITIZERS
As mentioned previously, radiotherapy is a common method of treating cancer that uses ionizing radiation to destroy tumor cells.
However, some cancers are resistant to radiotherapy because of tumor heterogeneity and biological changes. Cell cycle alterations, hypoxia, cancer stem cells, inflammation, and DNA damage repair systems are factors that influence tumor resistance to radiotherapy. 105,106 The degree of radiation density in each tissue depends on the interaction of that tissue with the incident X-rays, the density of electrons, and the amount of energy absorbed. 107 High-Z NPs can be designed to have photothermal, photoacoustic, and ionizing radiation absorption properties, while remaining chemically inert. 108,109 High-Z NPs have some attractive properties, including low toxicity, easy preparation, easy surface functionalization, controllable size and morphology, and good chemical stability. 110,111 The effectiveness of these NPs was first observed in head and neck cancer patients who happened to also have metal implants, and who then underwent radiotherapy. 112 Dose enhancement factor (DEF) is determined by dividing the deposited dose in the blank condition by the deposited dose amongst tumors harboring nano-radiosensitizer. The three primary variables regulating the DEF were linked to the materials and pharmaceutical attributes (e.g., atomic number), incident radiation factors (e.g., photon energy), and subcellular localization of the nano-radiosensitizer. 113 Nano-radiosensitizers featuring high-Z elements promote cancer therapies via three principal mechanisms: physical, biological and chemical enhancement. In comparison with water, high-Z materials As discussed earlier, increased electron number production brings an enhanced direct and indirect effect on cancer cells which is studied under biological effects. In the other words, biological effects indicate the role of nano-radiosensitizers on inducing oxidative stress, cell cycle modulations, bystander effects. 114 In addition to the ROS production through physical enhancement, superficial atoms of the nanoparticles could act as a catalytic platform by transferring electron to the molecular oxygen. 115 Due to the surface area increase followed by size reduction, gold nanoparticles with 3 nm size showed two-fold turnover frequency (TOF) over 30 nm. 116 Therefore, dense coating/ functionalization on nano-radiosensitizers restricts the chemical enhancement effect.
High-Z metal NPs increase the local dose and focal ionization in surrounding cells through the photoelectric effect. 117 The mechanism of how photons interact with high-Z NPs is strongly related to the energy of the radiation beam, because the photoelectric effect decreases when the photon energy increases. With photons in the keV range, these NPs can increase the local radiation dose by 10 to 150 times relative to the surrounding soft tissue. 118,119 Therefore, keV energy photons should be used in combination with these NPs to optimize the radiosensitization effects.
In photoelectric effect, the electron gets pulled out of the material if it receives a photon's energy and the photon has greater energy than the work function. Also, an atom emits a second electron when one of its internal electrons vanishes, a process known as an auger electron emission. In this effect, the second electron released is termed Auger electron. The process of the physical phenomena upon photon absorption, electron release and following DNA damage is represented in ( Figure 5). The photoelectric effect, which predominates at low photon F I G U R E 4 Schematically illustration of nano-radiosensitizers utilization in vivo and its cellular effects energy, may occur in subsequent to auger electron emission. In the other words, a cascade of low-energy electrons that move over short distances and deposit their energy locally is generated by auger electron emission. These electrons can directly interact with biomolecules or produce ROS. 120-122 Therefore, utilizing Auger electron for cancer treatment demands localization of nano-radiosensitizers near to the nucleus. 123 Using high-Z elements with relatively low energy radiation (tens to hundreds of keV) is a promising way to treat resistant cancers.
Loading the tumor with high-Z elements produces a differentiating effect, by increasing radiation dose to the tumor and reducing it to the surrounding healthy unloaded tissue. 124 On the other hand, photons in the keV range are generally not widely used in the clinic due to their low penetration depth. The low energy of Auger and photoelectric electrons is entrapped by other atoms of the nanoparticles. 125 Conventional photons used in clinical radiotherapy have an energy in the range of 6-20 MeV. 126 The probability of the photoelectric effect relative to the Compton effect and ion pair production decreases upon increasing the photon energy. Moreover, biological effects such as bystander, oxidative stress, and DNA damage can lead to radiosensitization with both MeV and KeV range photons. Interaction between radiation and high-Z NPs produces significant levels of ROS, resulting in oxidative stress, DNA damage, and apoptosis. 114,126 In addition, while most high-Z metal NPs cannot penetrate into the cell to affect the nucleus, but by increasing the physical dose, they can arrest the cell cycle, and DNA damage is caused by increased ROS production. 127,128 The targeting efficiency of high-Z metal NPs is also important to determine the overall radiosensitizing efficiency. Today, various high-Z NPs, such as hafnium oxide NPs, gadolinium oxide NPs, gold NPs, bismuth NPs, and silver NPs have all been studied for radiosensitization, each with their own unique feature. The next section will discuss the properties and function of these NPs.

| Hafnium oxide NPs
Hafnium (Hf) is an element with high Z (Z = 72) and an electron emission ability that is used to produce X-rays. 129 The properties of this element include remarkable plasticity (stretchability), hightemperature resistance, processability, and corrosion resistance. 130 The radiosensitization ability of hafnium oxide NPs has been reported in several in vivo and in vitro studies. 8 (Nanobiotix, France) is a commercial product made of 50-nm crystalline hafnium oxide NPs, functionalized by a negatively charged phosphate in aqueous solution (pH 6-8), which can be injected intratumorally and activated with external beam radiotherapy. [132][133][134] In addition, hafnium oxide NPs are inert and have no toxicity to living cells, which made them promising in various clinical trials. 135 The phase II/III trial showed positive results in patients with soft tissue sarcomas. 136 Studies showed that radiotherapy-activated NBTXR3 F I G U R E 5 Auger, photoelectric, and Compton electrons mediated direct and indirect DNA aberration in nano-radiosensitizers.
NPs also played an essential role in inducing an anti-tumor immune response. 134,137 Radiotherapy plus NBTXR3 NPs can not only enhance the cell death caused by standard radiotherapy but can also activate further pathways for tumor cell death and immune response activation.
Maggiorella et al. 107 injected NBTXR3 NPs into sarcoma bearing mice and then used cobalt 60 source radiation. They found that 24 h after NP injection and radiation, tumor growth was significantly inhibited compared with mice receiving radiotherapy alone. The crystal structure of these NPs did not change after a long time in vivo, which indicates the appropriate interaction of these NPs with ionizing radiation.
Hoffmann et al. 129  NPs were not excreted in the urine, and did not leak into the tissue surrounding the tumor, and no side effects were observed. This study showed that NP amounts >10% of the tumor volume had a beneficial response over time, and was reported to be suitable for elderly or chemotherapy intolerant patients. 129

| Gadolinium oxide NPs
Gadolinium (z = 64) is another element with a high z atomic number with eight unpaired electrons, a + III oxidation state, and high coordination numbers between eight and ten. 138 Gadolinium-based NPs have been used in radiosensitization, 139 neutron capture therapy, 140 and as a contrast agent in magnetic resonance imaging (MRI). 141 These nanoparticles are excreted in the urine within a few hours after intravenous injection (up to 30%). 142 Several factors such as the clustering and heterogeneous distribution of gadolinium atoms, the widespread distribution of low-energy electrons, and the optimal type of X-rays can cause the radiation to be concentrated at the site and the tumor cells to be killed. 124,143 Using these NPs, a lower radiation dose concentration can be delivered to the nucleus than to the cytoplasm or membrane. 124 Gadolinium-based NPs have beneficial properties, such as high relativity, high biodistribution, passive uptake in tumors, and high permeability. 144 Gadolinium interacts with various types of ionizing radiation, such as X-rays, gamma rays, neutrons, or electrons, which could benefit its rapid translation to the clinic. 145 Gadolinium-based NPs display biocompatibility and a stable chemical composition, but the type of radiation used will affect their performance. [146][147][148] The main limitation of utilizing these NPs is the similar ionic radius between gadolinium (III) and calcium (II), which leads to the potential replacement of calcium by gadolinium in bone. 149,150 In addition, the release of gadolinium could cause systemic nephrogenic fibrosis, therefore care must be taken in using these nanoparticles. 150,151 Motexafin Gadolinium (MGd) (Xcytrin, Sunnyvale, CA) is a compound of gadolinium that is used as a photosensitizer in photodynamic therapy for cancer, by producing ROS and inhibiting tumor growth. It also increases MRI signals, targets cancer cells (such as glioblastoma and brain metastases) and enhances cytotoxicity in combination with radiation. Motexafin gadolinium is currently being tested in clinical trials as a radiosensitizer. 152,153 Dotarem (Guerbet, France) is another commercial product based on gadolinium, which is used as a contrast agent in MRI. 154 The safety and efficacy of Dotarem have been demonstrated in more than 7000 patients. Dotarem excretion occurs by glomerular filtration in the kidneys, or by peritoneal dialysis.
In patients with renal insufficiency, Dotarem is slowly eliminated but still has good safety, however it can occasionally cause anaphylactic shock. 155 Mignot et al. 156

| Gold NPs
Gold nanoparticles have been extensively studied as tumor radiosensitizers due to their advantages including strong photoelectric absorption coefficient, good biocompatibility, and their high surfaceto-volume ratio. 10 Due to their enhanced permeability and retention (EPR) properties, gold nanoparticles can accumulate at the tumor site, but have low permeability to capillaries and normal blood vessels in other tissues, such as the heart. They can be used as an imaging contrast agent, in order to diagnose diseases and track treatments. Due to their controllable size, and unique chemical, electrical and optical properties, gold NPs have become a major candidate for use in biological applications. 10,126 In vivo research has shown that X-ray RT combined with gold NPs acting as a radiosensitizer, can increase the survival of tumor bearing mice. 162 Radiation plus gold NPs increased the formation of free radicals in cancer cells, and interrupted the cell cycle. 114 The lethal effect of NPs as radiosensitizers depends on their size, while according to a previous report, gold NPs with a diameter of about 13 nm coupled with a radiation dose of 4 to 8 Gray (Gy) may have the best lethal effect. 7,10,126 Gold NPs at a dose of 6 Gy provided the most lethal effects to inhibit tumor growth. 102 Gold NPs larger than 30 nm showed the same effect as 13 nm, but at the same time, their toxicity was higher. 102 Nanoparticles coated with polyethylene glycol (PEG) with a diameter of $13 nm have been used to improve CT scan imaging and radiosensitivity with optimized results. Moreover, gold NPs can also be modified with several ligands to allow drug and gene therapy, and increase the biocompatibility of these nanoparticles. 102

| Bismuth NPs
Since bismuth has a high atomic number, low toxicity, and low cost, bismuth nanoparticles could act as diagnostic and therapeutic agents, while they have also attracted widespread attention as a design factor in radiation therapy and imaging. 163 Bismuth-based sensitizers have low toxicity, easy availability of NPs, and cost-effectiveness as their advantages. 164 As a biocompatible element with an atomic number of 83, bismuth can maximize the efficiency of radiation absorption, and has been clinically used for many years. Bismuth NPs have biodegradable properties and can be removed from the body as soluble ions. 165,166 Various synthetic methods have been developed to increase the efficiency of bismuth NP preparation, including thermal dissolution, photochemical, and precursor methods. [165][166][167] Recently, folate-modified bismuth NPs coated with erythrocyte membranes have been developed for breast cancer radiotherapy, especially to increase the generation of free radicals. In addition, cellulose nanofibers have been used to fabricate bismuth NPs, which increased the production and secretion of free radicals in the presence of X-rays, resulting in good tumor destruction.
Due to the presence of carbonyl groups on the bismuth nanofibers, they were effectively absorbed and local oxidation was prevented, making them biocompatible. 165

| Silver NPs
Silver NPs can kill cells by apoptosis, activation of oxidative stress, and induction of excessive membrane fluidity. 102 Silver NPs have unique optical, electrical and antibacterial properties, and have been widely used in biosensors, photonics, electronics, and antimicrobial applications. The investigation of silver NPs in cancer treatment has yielded positive results. 168,169 The use of silver NPs as radiosensitizers, especially in the treatment of brain tumors, has been investigated with promising results. For example, silver NPs showed a better radiosensitizing effect versus gold NPs (at same molar mass) on glioma tumors, leading to increases in autophagy and apoptosis. 170 However, hybrid combinations of silver NPs with other types of NPs can strengthen the sensitizing effect. [171][172][173][174][175] In order to combine the different properties of separate NPs together, the easiest way is to coat them both in a suitable shell, such as silica. 176

| QUANTIFYING RADIATION SENSITIZATION TECHNIQUES
The use of NPs changes the quality of radiation and creates complex patterns of ionization, which ultimately leads to fatal damage to cells. 177 Multiple reports show the effect of NPs on cell cycle, metabolic activity and DNA repair pathways. These effects depend on a complex range of physical, chemical, and biological parameters of NPs such as size and type of material, charge, coating, reactive radical production and cell uptake rate, cell cycle, etc. 114 On the other hand, the most widely used method in radiobiology to study the effectiveness of a treatment is the clonogenic method (or colony formation).
A cell that has lost its ability to reproduce is considered dead. This type of assay is the most widely used method to evaluate the radiation sensitivity of various cell lines and is considered as the gold standard for determining the response to radiation. 178,179 Despite numerous researches related to the use of NPs with radiation, there is a few precise and appropriate guide to evaluate the effectiveness of NPs. Having a comprehensive list of procedures and clear guidelines on comparative quantification methods used to assess radiation increased due to the fact that NPs application would ameliorate the NP and radiobiological community to better understand NPmediated effects and translate NPs studies to the clinical stage. 180,181 The number of colonies formed after treatment is calculated as a function of radiation dose and provides survival curves for evaluation. Primary antibodies target specific repair proteins that are conjugated by gold secondary antibodies similar to immunocytochemistry. 184 Although TEM has not been used to study DNA damage caused by radiation sensitivity, it was used to monitor cell uptake and distribution of NPs. 185 Furthermore, recently the technical use of TEM has been thoroughly described as a tool to study NP-induced radiosensitivity in vitro. 179,185 Furthermore, flow cytometry could be used to detect DNA damage and analyze the cell cycle. 186 Propidium iodide is the most commonly used dye for quantitative assessment of DNA content and is a very useful technique for studying various checkpoints during the cell cycle. 187 In this method, nucleotides were extracted from cells, then they were stained with ethidium bromide fluorescent dye, and then they were exposed to laser light in flow cytometry. 187 Flow cytometry and TEM are also used to investigate cellular uptake and final localization of nanoparticles. 188 189 This method can be an alternative method for agarose gel electrophoresis to analyze the formation of DNA fragments during apoptosis. 179,190 Immunoblot or western blot is also used to detect changes in protein expression after treatment. 191 In this method, protein expression is sampled at different time points after X-ray irradiation to determine how NP pretreatment increases radiation sensitivity. 191 This is done both in the presence and absence of NPs. Primary antibodies were considered based on apoptosis, DNA damage, repair and oxidative stress. 192 The ROS is one of the possible causes of aging, while radiation could lead to an increased senescence phenotype. Since ROS production could be responsible for NP-induced radiosensitization, it is necessary to investigate whether cells irradiated with NPs lead to increased senescence compared with cells irradiated alone. 193 In general, the radiation sensitivity effects of NPs could be classified into three groups, including physical, chemical, and biological effects. Table 2  can induce an immune response by releasing tumor antigens, which can be magnified even further in combination with immunotherapy such as checkpoint blockade inhibitors. [199][200][201] Another technology based on laser radiation is photodynamic therapy (PDT) which produces cytotoxic reactive oxygen species (ROS) causing cancer cell death. PDT can employ organic photosensitizers (e.g., porphyrins, phthalocyanines, or dyes) or inorganic photosensitizer agents (e.g., semiconducting NPs or quantum dots). In both cases a photochemical reaction takes place upon laser irradiation leading to the generation of ROS. 202 RT is a very common cancer therapy method all around the world.
In this method, the high energy radiation is irradiated to the tumor to produce ROS following ionization, which interacts with cellular compartments leading to death. 203 Recently, nanoparticle-based radiosensitizers have emerged for boosting RT in a targeted and precise manner. 127 The engineered NPs target the tumors and generate multiple electrons (e.g., Auger electrons, photoelectrons) upon receiving radiation. 114  can provide a similar boost to the immune response as RT. 208,209 Utilizing drug delivery approaches alongside RT has shown promise to treat cancer while minimizing the RT side effects. 210 In addition, nitric oxide delivery is a recently emerging approach in cancer therapy, which enhances the RT effect in hypoxic tumors via angiogenesis regulation 211 and a direct anticancer effect. 212 The synergistic effects of radiosensitizing NPs have been studied in combination with several approaches discussed below.

| Immunotherapy
The combination of RT and immunotherapy has recently been reviewed, 213  Major routes for immunosuppression include the programmed cell death protein 1 (PD-1) and its two ligands (PD-L1 and PD-L2). Moreover, researchers also used two-dimensional nanosheets (1.6 nm thickness) containing the hafnium MOF. 229 The MOF structures constructed via porphyrins (hafnium-5,15-di(p-benzoato) porphyrin, 5,10,15,20-tetra (p-benzoato) porphyrin, and etc.), produces singlet oxygens in addition to •OH, demanding a lower radiation dose for inducing abscopal effect. 230 Dong et al. 231 233,234 Also, NBTXR3 activates the cGAS-STING pathway which facilitates the IRF3/7 transcription factor entrance to the nucleus and type-1 interferon (IFN-1) release thereof. 131,235 As higher dose of radiation may trigger cytosolic DNA degradation via TREX-1 exonuclease, using a proper radiation dose is demanded in order to leverage STING pathway mediated immunotherapy. 236 In order to enhance the radioimmunotherapy response, utilizing the capability of polymeric based nanoparticles is addressed. Patel et al. 237 used bacterial membrane coated polyplex nanoparticles containing PC7A (pH-responsive polymer) and CpG (TLR9 agonist). In these nanoparticles, the released neoantigens following RT were trapped by maleimide modification of the NPs and transported toward dendritic cells for the next immunologic steps. The maleimide functional groups captured the neoantigens by forming thioester bonds. 238 In a study by Pang et al. 239 polysaccharide NPs extracted from Astragalus membranaceus natural herb were used to inherently activate DCs.

Immunosuppressive mechanisms governing T cells are inhibited in
These nanoparticles inhibited both primary and secondary tumor growth in combination with RT, which agreed with the increased populations of CD4+ and CD8+ T-cells. These advantages led to a robust antitumor immune response in combination with checkpoint blockade inhibitors. 228 Chen et al. 240

| Gene and nucleic acid delivery
Combining gene therapy approaches with RT may be a promising strategy to overcome tumor resistance to radiation, and increase the therapeutic response to RT. 243 The use of radiation inducible promoters for genes involved in cell cycle checkpoints, cellular stress, DNA repair, and apoptosis could lead to more effective and specific RT, and reduce off-target effects. 243 Kaliberov et al. 243  Given the complex physiology of tumor cells and their various tumor escape mechanisms, combination therapies often result in better responses. However, using several therapeutic approaches together could also increase the treatment side effects. 243,245 As mentioned before, specific approaches could be used to improve the sensitivity of tumor cells to RT. Yang et al. 246 have designed zwitterionic Au-containing dendrimers to deliver hypoxia inducible factor-1α (HIF-1α) silencing siRNA while providing radio-enhancing effect. Knockeddown HIF-1α plus radiation have decreased the metastatic behavior through downregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9) expression. Although radiosensitizing NPs and gene delivery have been used separately for sensitizing tumor cells to radiation therapy, 243,245 however, the publications on their combination are scarce. Undoubtedly, targeting multiple pathways with gene therapy approaches coupled with radiosensitizing NPs could be a promising future strategy.

| Photothermal therapy (PTT)
The success of radiosensitization approaches is highly dependent on the type of tumor that is targeted. There are three primary types of DNA lesions involved in RT: SSB, DSB, and damage to nucleic acid bases. 247  The effect of hyperthermia is highly dependent on the phase of the cell cycle. Accordingly, cells in the M and S phases of the cell cycle are more vulnerable toward hyperthermia. 251 In contrast, the G phase is resistant to heat treatment, which should be taken into account because radiation is more effective with cells in the G2/M phase. 252 The poorly developed vasculature of the tumor leads to an oxygen-deprived and acidic microenvironment, which diminishes the efficacy of RT. Hyperthermia at high temperatures directly kills the cells, even within a hypoxic environment through unclear mechanisms. 253,254 However, direct cell killing at low temperatures under hypoxic conditions requires a more prolonged cycle of hyperthermia. 255 In addition, cells treated with hyperthermia express HIF-1α, which switches the cellular metabolism into glycolysis, leading to diminished oxygen consumption. 256,257 Moreover, hyperthermia has been shown to be promising in some clinical trials to enhance the efficacy of RT. 258,259 The effect of in vivo hyperthermia at mild temperatures (40-42 C) may lead to vasodilation, and therefore greater oxygen delivery to the tumor, thus sensitizing the cells to RT. 260 Drainage of the interstitial fluid under elevated temperature is considered proof of increased oxygenation. 261 Obtaining uniform distribution of the heat over the entire heterogeneous structure of the tumor is the main hurdle against hyperthermia-mediated therapy. 262 Table 3 summarizes some studies on the combination of PTT and NP radiosensitizers. A study by Li et al. 267 showed that PTT prior to

| Photodynamic therapy
Photodynamic therapy (PDT) is an emerging technology for treating cancer, with some clinical approvals in recent decades. In this method, the light is aborbed by a photosensitizer (PS) and initiates a photochemical reaction leading to ROS generation by two mechanisms, types I and II. In type 1, electrons are transferred from the excited triplet state PS to adjacent biomolecules to form radical cations and radical anions, that go on to form ROS. 272 In type 2 PDT the excited PS interacts with surrounding molecular oxygen to form the extremely and direct electron (Auger) generation. 278

| Oxygen delivery
Oxygen is an essential component of RT, and increases the overall efficacy by improving ROS generation within the tumor. The earlier drugs used for improving the oxygen level of the tumor microenvironment were discussed previously. Oxygenation of the tissue using biomaterials, is often carried out by loading molecular oxygen into rationally designed formulations, which then produce free oxygen following dissociation.
Perfluorocarbon (PFC)-based materials have been clinically used as artificial blood substitutes, due to their high gas dissolving capacity and chemical inertness. 279 Certain compounds (sodium percarbonate, hydrogen peroxide, and calcium peroxide 280 ) can generate oxygen when they come into contact with water, or are catalyzed by catalase. Beyond the delivery of physically entrapped molecular oxygen, NPs with inherent oxygen generation capability can operate by water splitting, or by converting tumor-residing hydrogen peroxide into oxygen. 281 NPs can be incorporated with PFCs to deliver more oxygen for sensitizing tumors to RT. 282 Song et al. 283 fabricated PEG-modified hollow Bi 2 Se 3 NPs, which served as a reservoir for PFC delivery and thermo-radiation therapy of the tumor. Active oxygen delivery was demonstrated by an accelerated rate of oxygen release rate upon laser irradiation. The same group reported a higher PFC loading, using a PFC nanodroplet hybrid with high-Z tantalum oxide (TaOx) (TaOx@PFC-PEG) nanoparticles. This system was able to considerably increase RT efficacy owing to its multifunctional activity. 284 Ultrasound-triggered oxygen release was reported by Jiang et al. 285 using hierarchical multiplexed nanodroplets of liquid perfluorooctyl bromide and ultrasmall gold NPs as an oxygen carrier and radiosensitizer, respectively. The NPs were able to prevent unwanted DNA DSB repair by delivering oxygen to the cells, as shown in Figure 8a. To prove the concept, the γ-H 2 AX content (molecular marker of DNA damage and repair) was measured at different time intervals post-Xray irradiation (5 Gy) (Figure 8b, c).
Other method for oxygenating the tumors is to utilize the inherently accessible hydrogen peroxide and convert it to the molecular oxygen through catalytic reaction. Therefore, NPs bearing catalaselike activity could be leveraged for oxygen-feeding. Namely, Porphyrin-based MOFs decorated with gold nano-radiosensitizer and hafnium-based manganoporphyrins increased the oxygen level up to 6 and 23 mg/L, respectively, in presence of H 2 O 2 . 225,286 Cyanobacteria are considered as the initial oxygen generator on the earth utilizing the following reaction 287 : Different strains of cyanobacteria are able to produce nutrients and remove the unwanted compounds from the medium. 288 Amongst with, Synechococcus elongatus has been used in vitro and in vivo for supplying the molecular oxygen for ischemia 289,290 and PDT. 291,292 Chai et al. 293

| Nitric oxide delivery
Nitric oxide (NO) is a naturally occurring free radical gas produced by bacteria and mammals, which plays a significant role in many cellular signaling pathways. 306,307 NO is synthesized by the transformation of L-arginine to L-citrulline by the isoforms of nitric oxide synthase (NOS). 308 Initial studies on the role of NO in cancer therapy showed its over-expression in several cancers. 309 Since then, several studies have investigated its role in cancer and possible ways to fight cancer, in a similar manner to ROS. 310 According to the levels of NO, its effects are classified as direct reactions (<200 nM) and indirect reactions (>400 nM). 311 Direct reactions occur immediately upon NO release, which involve direct interactions with biological receptors. Indirect reactions refer to NO reactions with oxygen or superoxide, which then produces reactive nitrogen species (RNS). 312 Researchers have used NPs to produce intracellular NO in some photoinduced approaches [313][314][315] and multimodal therapies. 316,317 NO is a well-known substance which can increase tissue oxygenation by dilating the tumor blood vessels by several routes. [318][319][320] In contrast, the use of nitric oxide synthase inhibitors, such as NG-nitro-L-arginine (L-NNA), can inhibit anti-apoptotic pathways 321 and decrease tumor blood flow leading to chronic hypoxia, cancer cell death, as well as protection of normal cells under radiation. 322,323 Additionally, NO is an important player in the bystander effect, owing to its rapid diffusion across cell membranes. The bystander effect describes the destruction of tumor cells adjacent to (but outside) the irradiated zone by the generation of toxic molecules from the irradiated cancer cells. 324 In a study by Han et al. 325 they found that cells that were physically separate from others receiving α-particle irradiation formed γ- Ren et al. 335  The time-consuming coating procedure and sampling from the patient in clinics will make this targeting method hard to translate in clinics.

| PTT/PDT/RT
Due to the inability of RT to destroy massive tumors, the combination of multiple therapies may be preferable. Meanwhile, PTT has shown promise as an adjunct therapy in clinical studies due to the abovementioned synergistic pathways. 258 Adding PDT to therapies that utilize photonic sources to produce ROS along with radiosensitization is logical.
To this end, Qiu et al. 371  In conclusion, combination therapy involves numerous different techniques that can be used together for cancer treatment, while also minimizing conventional therapeutic resistance. However, accurate understanding of the mechanisms underlying combination therapies is essential for accelerating their clinical translation. When combination therapy is compared with the constitutive methods used alone, the best combination of modalities may be selected. 374 Recently emerging laboratory methods, such as organoid culture models may be used to recapitulate tumor-associated components and uncover hidden effects. 375

| CONCLUSIONS AND PERSPECTIVES
The emergence of high-Z NPs as radiosensitizers with multifunctional properties is expected to revolutionize conventional radiation therapy.
Elucidation of the exact mechanisms behind each therapeutic modality, while understanding their synergistic effects is crucial for their clinical translation. Insufficient knowledge about the role of photothermal therapy as an immunological mediator has restricted our deeper insight into radiation and photothermal combination therapy mechanisms.
Despite some successes, research on the combination of radiosensitizers and newly introduced therapeutic modalities, such as sonodynamic therapy and gene delivery has remained scarce. The delivery of oxygen and novel methods for in situ oxygen production through catalytic reactions, have been recently explored to boost RT efficacy.
However, the use of PFC-based compounds, regardless of their drawbacks, could accelerate the clinical translation.
The discovery of the best combination of modalities and their many variable parameters (e.g., duration, dosage, and sequence of administration) is challenging to design trials for eventual clinical application. The use of tumor mimicking laboratory technologies such as spheroids, microfluidics, and artificial intelligence could all be beneficial to tackle the aforementioned problems.
Theranostic agents can combine both multiple therapeutic effects as well as imaging modalities into a single system. These can provide on-demand tracking of the NP biodistribution. Nano-radiosensitizers often show high X-ray attenuation which could be utilized as CT-scan imaging contrast agent. High gap amongst k-edge of high-Z nanoparticle and biological tissue enables photon counting CT imaging. Engineering nano-radiosensitizers could add additional MRI imaging modality via surface modification (e.g., with Motexafin Gadolinium), doping (e.g., with Mn and Gd) and heterostructures (e.g., with SPIONS). Also, quantum dots based on high-Z materials with fluorescence imaging capability could be used as theranostic agent.
Immunotherapy and radiotherapy have been used clinically, however, they suffer from low efficiency on several metastatic cancers or dense tumors. Amongst studied combination, adjuvant effect of nanoradiosensitizers in immunotherapy has more potential to be used in

FUNDING INFORMATION
This study was not funded by any funding institute. Michael R. Hamblin was supported by US NIH grants R01AI050875 and R21AI121700.

CONFLICT OF INTEREST
Michael R. Hamblin declares the following potential conflicts of inter-

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.