Radiotherapy utilizes high-energy beams to destroy cancer cells and thereby shrink tumors [39]. On the other hand, chemotherapy (CT) uses cytotoxic drugs that are capable of killing cancer cells and inhibit cancer cell growth [40]. These conventional treatments have limitations, such as insufficient therapeutic properties and side effects [41]. In this regard, combination of RT with CT offers an improved therapeutic effect compared to using a single approach. CT is widely used in lung cancer [42], esophageal cancer [43], rectal cancer [44], and hepatocellular carcinoma [45]. However, toxic side effects and a lack of selectivity and synergy between RT and CT are key problems in chemotherapeutic treatment. In addition, severe side effects of Pt-based anticancer drugs have restricted their clinical application [41,46]. On the other hand, RT is often off-target and damages surrounding healthy cells, and it is difficult to obtain optimum radiation [47]. The application of nanostructured radiosensitizers in RT has attained great attention recently. Using high Z materials as nano radiosensitizers enhances RT owing to their Compton scattering and photoelectric effects [48]. Moreover, introducing platinum-based anticancer drugs, such as cisplatin (II), oxaliplatin (II), and carboplatin (II), as radiosensitizers has yielded more effective RT strategies [49].

A key advantage to combined treatment is the prospect of achieving a greater organ-preservation rate. Another advantage is its independent cell-destroying effect. RT aims to control the primary tumor, whereas CT eliminates distant metastasis. The combined treatment modality is also advocated based on clinical trial results. Phase II trials have reported convincing results of combined treatments [50]. However, the combined approach has some challenges to overcome. Several parameters, such as the dose, duration, administration sequence, should be optimized properly [51]. Therefore, further research is needed to explore these limitations.

The bombarding of ionizing radiation with NPs gives rise to several outcomes, including photoelectric effects, the Compton electron effect, and Auger electron effects (Figure 1) [40]. The radiation energy is imparted to the electrons of the NPs’ atoms, causing the ejection of electrons from their orbits [41]. Such electronic ejection occurs with a kinetic energy that is equivalent to the radiation wave energy minus the binding energy of the electrons and assesses the electron range in tissue [42].

Photoelectric effects occur when low-energy photons interacts with materials (<60 keV). The photon energy is exclusively absorbed by inner orbital electrons and is ejected from its orbit. This phenomenon causes the electrons of outer orbits to shift to inner orbits and empty space. Thus, liberated fluorescence photons with specific wavelengths depend on the difference between the energy of two orbits, called secondary radiation (Figure 1). Later, Auger electrons are emitted when outer orbit electrons fill the empty space of the inner orbit due to photoelectric effects. This process relinquishes energy to outer orbits electrons, leading to ejection of electrons from higher orbits (Figure 1) [17]. The Auger electrons have high linear energy transfer properties and hence could be extremely injurious to cells [43]. The probability of photoelectric effects occurring is assessed by the formula (Z/E)³, where E = the incoming photon’s energy, and Z = the absorber molecule’s atomic number. Thus, the possibility of photoelectric effects is enhanced with increased absorber atoms, but it decreases with increased energy of incident photons. Photoelectric effects contribute more to radiation interaction with high Z metal NPs than to absorption in soft tissue. Therefore, photoelectrons, secondary photons, and Auger electrons released from high-Z metal NPs enhance localized doses, along with focal ionization of nearby cells via photoelectric effects. Since photoelectric effects decrease with increased energy of photons, most of the nanoparticles combining with radiation treatment use keV photons to optimize the radiosensitization and enhance the local dose by 10–150 times [44,45,46].

The Compton interaction dominates within 25 keV–25 MeV of photon energy. Since most RT is performed at an energy level of 6–20 MeV, this effect is the most common interaction between incident photons and cancer tissue. In the case of the Compton effect, incident photons strike weakly bound outer orbit electrons and donate part of their energy to the electrons, stimulating electrons to leave the outer orbit. Concurrently, the photons become scattered after giving part of their energy and further interacting with other atoms (Figure 1). Afterward, the emitted electrons continue to ionize adjacent tissues. The possibility of a Compton interaction depends inversely on the incoming photon’s energy but is not dependent on the material’s atomic number. Therefore, high-Z metal NPs do not have a substantial role in the Compton effect [17,40,46,47].

Complementing nanotherapeutics with ionizing radiation exhibits an enhanced biological response in cancer management through several approaches. The major mechanisms include inhibiting DNA-repair processes, producing reactive oxygen species (ROS), which damage DNA or other biomolecules by oxidation, inhibiting tumor metastasis by controlling the tumor microenvironment (TME), and arresting the cell cycle (Figure 2).

Ionizing with X-rays, γ-rays, or proton radiation itself causes spontaneous double strand breaks (DSBs) in DNA by breaking atomic and molecular bonds [48,49,50], and un-repairing of DSB leads to genetic instability, cell division termination, and reduced proliferation and consequently to death [51,52]. However, such DSBs are repaired by the cellular DNA damage response (DDR) [53]. Three main proteins from the phosphatidylinositol 3-kinase-related kinase family, namely ataxia-telangiectasia mutated (ATM), ATM and Rad3 related (ATR), and DNA-dependent protein kinase (DNA-PK), are involved in identifying and repairing DNA DSBs [54]. Three different types of sensor protein complexes are responsible for the recruitment, as well as activation, of these three proteins of the PI3K family at damaged DNA sites, i.e., MRE11/RAD50/NBS1 (MRN) for ATM, RPA/ATRIP for ATR, and KU70–KU80/86 for DNA-PK [55].

The main goal of DDR machinery is to delay the progression of the cell cycle and fix the damage [56]. When cancer cells are exposed to IR, they undergo transient cell cycle arrest to repair DSBs either by non-homologous end joining (NHEJ) throughout the entire cell cycle or by homologous recombination (HR) during the S and G2 phase [57]. ATM is primarily responsible for activating the HR pathway. Other proteins, including breast and ovarian susceptibility protein (Brca2), Rad51, and X-ray repair cross complementing protein 2 (XRCC2), are also involved in the HR pathway [58]. On the other hand, DNA-PK mainly regulates the NHEJ pathway in association with DNA ligase IV and X-ray repair cross complementing protein 4 [59,60]. Another pathway involves both ATM and ATR. Here, MRN sensor protein complex senses damage sites and activates ATM [61]. Autophosphorylation of ATM kinase sends signals to transducers such as checkpoint kinase 2 (Chk2) and the transcription factor p53. p53 controls the expression of p21, which interacts with cyclin-dependent kinase (CDK) complex and arrests the G1 phase of the cell cycle [62]. Modification of chromatin also occurs together with the process, and then the DNA repair process is initiated. However, mutation of the p53 makes the G1 checkpoint defective in most of the cancer cells. Hence, the G2 checkpoint plays a crucial role for surviving cancer cells. RPA/ATRIP sensor protein complex recognizes and activates ATR. Here, ATR phosphorylates checkpoint kinase 1 (Chk1), which degrades cell division cycle 25A (CDC25A) through further phosphorylation and slows the progression of DNA replication during the S phase. The ATM-Chk2 and ATR-Chk1 signaling pathway acts together with DNA-PK phosphorylate p53, which controls genes required for DNA repair, arresting the cell cycle, and apoptosis [55,61,63].

For this purpose, DNA double-strand repair inhibition (DSBRI) appears to a promising strategy for RT [64]. However, it is a challenge to achieve tumor selective DSBRI-based radiotherapeutic treatment since such approaches often sensitize normal cells [65]. Interestingly, introduction of NP-based radiotherapeutic approaches mediates tumor-specific DSBRI owing to their increased permeability and retention effects [66,67]. Zhang et al. developed nano-constructure by combining androgen receptor (AR) with shRNA and folate-targeted H1 nanopolymer (NP AR-shRNA). NP AR-shRNA selectively destroyed prostate cancer cells by mimicking DNA DSBs and activated kinase activity, in turn impeding DNA damage repair signaling pathways (Figure 2) [68]. The presence of γ-H2AX is used as an indicator to detect DNA DSB in higher eukaryotes [69,70]. NP AR-shRNA in combination with IR (4 Gy) increases the expression of γ-H2AX in PC3 and 22Rv1 cells by nearly three-fold compared to IR alone (4 Gy). In vivo experiments showed that irradiation downregulated the expression of AR protein while increasing γ-H2AX expression in 22Rv1 tumors. Additionally, mice bearing PC3 and 22Rv1 cells were exposed to NP AR-shRNA under X-ray (total 9 Gy) and displayed significant tumor reduction compared to only X-ray treatment [68]. Similarly, Yao et al. formulated nanoparticles by conjugating DSB bait (Dbait) with H1 polymer (Dbait@H1 NPs), selectively killing prostate cancer cells upon exposure to radiation by inhibiting DSB repair [71]. Dbait@H1 NPs attached selectively to folate receptor and mimicked DNA DSBs upon release into the nucleus [72,73]. Dbait of Dbait@H1 NPs activates DNA-PK and phosphorylate γ-H2AX. Then, factors associated with DNA damage repair are assembled at the free end of Dbait, preventing them from affecting the DSB sites of the real chromosome and resulting in prolonged defects in DSB repair [74,75,76,77]. Irradiating (4 Gy) PC-3 cells with Dbait@H1 NPs induced γ-H2AX foci numbers three times higher than radiation alone (4 Gy). Moreover, the same radiation dose also increases phosphorylation of DNA-PK and H2AX of both PC-3 and 22Rv1 cells. Moreover, an in vivo study showed that mice carrying both PC-3 and 22Rv1 cells were exposed to 9 Gy of radiation and 60 μg/kg of Dbait@H1 NPs and exhibited 1.67- and 2.5-fold reduced tumor volumes, respectively, compared to only radiation (9 Gy) [71]. Similarly, HeLa cells exposed to AuNPs and irradiation at 4 Gy of 220 kVp and 6 MVp caused enhanced γ-H2AX, suggesting induction of possible DNA DSB [78]. Combined treatment with PEGylated-AuNPs and 4 Gy RT (150 kVp) increased DNA damage 1.7-fold in U251 cells compared to radiation alone [79]. Additionally, Zheng et al. reported that AuNPs at a radiation dose of 6 Gy induced DSB in HepG2 cells [80].

Chen et al. modified AuNPs with bovine serum albumin (BSA@AuNPs), which induced a 2.02-fold increase in γ-H2AX density compared to X-ray radiation only in U87 cells upon exposure to a 3-Gy dose of 160 kVp X-ray. Moreover, treating mice with BSA@AuNPs under X-ray radiation (5 Gy) reduced tumor volume significantly compared to X-ray radiation alone [81]. Similarly, a nanoformulation of KU55933 (NPs@KU55933) impeded the repair process of DSBs and exhibited enhanced tumor volume reduction in vivo.

Ionization in combination with NPs brings about indirect necrosis or apoptosis via oxidation of biomolecules, including proteins, lipids, and DNA, along with mitochondrial dysfunction [7,83,84]. High-intensity ionizing radiation generates ROS, including superoxide anion radicals (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (^•OH), through water radiolysis, and they interact with cellular biomolecules, leading to apoptosis/cellular death [7,85,86,87,88,89], as well as suppressing tumor progression [90]. Therefore, sufficient ROS production is crucial to mediate DNA damage, as well as suppress DNA repair (Figure 2).

Zhao et al. fabricated Gd-bearing polyoxometalates linked with chitosan nanospheres and integrated with hypoxia inducible factor 1α (HIF-1α) siRNA (GdW10@CS_HsiRNA) that showed enhanced radiosensitization in hypoxic tumors. GdW10@CS functions as an external radiosensitizer for depositing ionizing radiation doses and as a nanocarrier of HIF-1α siRNA to stop DNA DSB restoration. Additionally, GdW10@CS annihilates intracellular reduced glutathione (GSH) levels upon exposure to X-ray radiation, leading to overproduction of ROS through W6⁺-triggered GSH oxidation, thereby facilitating radiotherapeutic efficiency. Irradiating BEL-7402 cells with 6 Gy of X-ray and GdW10@CS (100 μM) produced Compton and Auger electrons, which interact with surrounding H₂O or O₂ molecules, thus producing 10-fold more ROS, as well as exhausting GSH levels three times more compared to X-ray treatment alone. Furthermore, 20 μL of GdW10@CS along with 10 Gy of X-ray radiation were administered into BALB/c mice bearing BEL-7402 tumors and reduced tumor volumes by nearly five- and eight-fold compared to GdW10@CS_HsiRNA without RT and RT without GdW10@CS_HsiRNA, respectively [91]. Zhan et al. designed a nano-enabled coordination platform with bismuth and cisplatin prodrug (NP@PVP) that improves the efficiency of chemoradiotherapy by X-ray radiation. The bismuth in NP@PVP increases generation of ROS to intensify DNA damage after X-ray irradiation. Treating EMT-6 cells with NP@PVP and X-ray irradiation (5 Gy) generated 3.21-fold more ROS compared to platinum-based drugs along with X-ray irradiation at the same dose. In vivo results showed that mice carrying EMT-6 tumors treated with NP@PVP (2 mg kg⁻¹) under irradiation with X-rays (5 Gy) showed 54.7% inhibited tumor growth compared to X-ray treatment only [92]. Choi et al. synthesized radiation-responsive PEGylated gold nanoparticles containing dihydrohodamine 123 (DHR-123) (RPAuNPs). RPAuNPs absorbs the X-ray energy and transfers it to nearby molecules through electrons, including photoelectrons, Compton election, and Auger electrons, which generate ROS by water radiolysis. Exposure of RPAuNPs (7.75 μg/mL) at to Gy of X-ray radiation increases the fluorescence by 7-fold owing to the local generation of ROS. The same treatment of MDA-MB-231 cells yields similar results, with increased ROS levels in nearby cells. Mice bearing MDA-MB-231 xenografts treated with RPAuNPs at a concentration of 6 μg/100 μL and irradiation of 6 Gy at 225 kVp exhibited 3- to 6-fold higher ROS production compared to the treatment with RPAuNPs without X-ray irradiation [93]. In addition, mitochondria are an important source of cellular ROS production [94]. Tang et al. constructed Gd-doped titanium dioxide nanosensitizer (G@TiO2 NPs), which targets mitochondria for effective RT. G@TiO2 NPs generates ROS effectively since it possesses a large photoelectric cross-section for X-rays [95].

The tumor microenvironment (TME) consists of various types of cells (endothelial cells, immune cells, fibroblasts, etc.) and extracellular components (extracellular matrix, growth factors, cytokines, etc.) that surrounds tumors and are nourished by blood and the lymphatic vascular network [96,97]. The TME functions critically in the regulation of tumor progression, immune escape, and metastasis [98]. It has significant influence on therapeutic effectivity [99]. Therefore, TME-associated NP-based RT offers potentiality in destroying cancer cells efficiently [100].

ROS are among the key players to alternate the tumor microenvironment (TME) during RT [101]. Among many other features of the TME, hypoxia mostly compromises tumor sensitivity to anticancer drugs, along with reactivity toward free radicals, thus creating hurdles in RT [7]. The anoxic and hypoxic TME of solid tumors compromises the production of ROS, causing poor responses to tumor cells [102,103]. Furthermore, hypoxia=activated hypoxia inducing factor-1 promotes resistance to RT and increases expression of genes involved in angiogenesis and metastasis of tumors [104,105,106]. Therefore, management of the TME is critical in killing cancer cells.

O₂ is indispensable during RT since it reacts with DNA breaks to avoid repair of DNA by tumor cells, thus relieving hypoxia and enhancing RT-mediated cell killing [107]. Considering this point, Liu et al. prepared a nanostructure system, PFC@PLGA-RBCM, by enveloping perfluorocarbon (PFC) within poly(d,l-lactide-co-glycolide) (PLGA), which then was coated with a red-blood-cell membrane (RBCM). PFC@PLGA-RBCM NPs contains a PFC core, which is able to dissolve O₂ to a great extent, and the RBCM coating contributes to enhanced blood circulation of NPs. PFC@PLGA-RBCM NPs deliver oxygen efficiently to the TME, which helps to relieve tumor hypoxia and enhance the efficacy of RT. Injecting 4T1-tumor-bearing mice with PFC@PLGA-RBCM NPs (200 µL) and exposing them to X-ray radiation (8 Gy) showed enhanced tumor volume reduction by nearly 8- and 2.5-fold compared to PFC@PLGA-RBCM NPs without irradiation and X-ray irradiation only, respectively [108]. Chen et al. fabricated NPs via encapsulating catalase (Cat) by poly(lactic-co-glycolic) acid and hydrophobic imiquimod (Cat@PLGA_R837). Moreover, X-ray irradiation induced gold NPs with silica cores (SAuNPs) to exhibit enhanced antitumor effects under a hypoxic environment. At 8 Gy of radiation, SAuNPs caused 20% more cellular death of CT26 cells than in a control group under hypoxic conditions, while only 5% death occurred under normoxic condition. In addition, irradiation with X-rays increased ROS production by 40% in normoxic conditions compared to 20% in hypoxic conditions [109].

Targeting the cell cycle is an effective approach in cancer treatment. Different nano formulations in combination with radiation mediate disruption of the cell cycle, leading to apoptosis [85]. Furthermore, cell cycle phases have distinctive effects on radiosensitivity. For instance, the late S-phase is the most radioresistant, while G2 is the most radiosensitive phase [110]. Cells activate cell cycle checkpoints in the G1, S, and G2 phases in response to radiation to repair genomic defects, maintenance integrity, or prevent cell division through activation of cell death mechanisms [111]. The literatures has reported that NPs with radiation arrest mostly the G2/M phase while reducing cells in the G0/G1 phase of the cell cycle (Figure 2) [112,113,114].

Roa et al. constructed gold NPs capped with glucose (Glu-AuNPs), showing improved cell-targeting capacity and excellent radio-sensitization. Glu-AuNPs stimulated activation of cyclin dependent kinase (CDK), leading to an accelerated G0/G1 phase and halting the G2/M phase of the cell cycle by activating CDK1 and CDK2. Irradiating DU-145 cells with 2 Gy of ortho-voltage together with Glu-AuNPs (15 nM) arrested the cell cycle at the G2/M phase and exhibited enhanced growth inhibition by 1.5- to 2-fold compared to X-ray alone. Glu-GNPs inhibited cyclin A’s expression by 42.3%. Cyclin A together with CDK2 form cyclin A–CDK2 complex and initiate the transition of G2/M. Hence, inhibiting cyclin A caused G2/M transition delay [115]. Chen et al. synthesized ultra-small selenium NPs (SeNPs) of 27.5 nm by chemical methods [116]. SeNPs have shown excellent biological activity and low toxicity [117,118]. Upon irradiation, SeNPs arrested the G2/M phase while accelerating the G1/S phase. X-ray irradiation of SeNPs (0.15 μg/mL) at 6 Gy on MCF-7 cells upsurged the G2/M phase proportion by 7.4-fold compared to radiation alone [116]. Xu et al. conjugated gold with glycine (G), arginine (R), and aspartate (D) peptides (Au-G-R-D). While exposed to 6-mV X-rays with a 4-Gy dose, Au-G-R-D (50 μg/mL) arrested the G2/M phase significantly (6.4%) in A375 melanoma cells compared to radiation alone [119].

Cancer stem cells (CSCs) are common in most cancers; they cause metastases and function as a cancer cell reservoir, causing tumor relapse after CT, radiotherapy, or surgery [151]. CSCs’ ability to proliferate unlimitedly and their resistance to drugs pose great threats in cancer management [152]. CSCs are characteristically resistant to CT due to their quiescence, capacity to repair DNA, and ABC-transporter expression [153]. The self-renewal property allows them to extend tumor cell numbers after chemo- or radiotherapy [154,155]. Conventional treatments are not able to destroy CSCs; therefore, novel treatment strategies are highly demanded.

Fiorillo et al. developed graphene oxide (GO)-based nanostructures that were able to prevent tumor-sphere formation in six different cancer cell lines, including MCF7 for breast cancer, SKOV3 for ovarian cancer, PC3 for prostate cancer, MIA-PaCa-2 for pancreatic cancer, A549 for lung cancer, and U87-MG for brain cancer. They applied tumor sphere assay to measure the formation of tumor spheres to evaluate the effect on GO. The results suggested that GO targets the phenotypic prosperity of CSCs and reduces bona fide CSC numbers by inducing their differentiation and inhibiting their proliferation. More specifically, GO-based treatment inhibited several major signal pathways, including WNT- and Notch-driven signaling, STAT1/3 signaling, and the NRF2-dependent anti-oxidant response, together with inducing the differentiation of CSC, thus decreasing the general stemness [156]. Likewise, Yao et al. designed gastric CSCs targeting carbon nanotubes based on chitosan and loaded with salinomycin with hyaluronic acid (SWCNTs), which selectively eradicate gastric CSCs [157]. Later, Al Faraj et al. modified SWCNTs with CD44 antibodies and showed enhanced targeting of breast CSCs and promise in clinical studies [158].