satellite ternary heteronanostructure for multimodal imaging-guided synergistic cancer radiotherapy

Journal Pre-proof Radiosensitive core/satellite ternary heteronanostructure for multimodal imagingguided synergistic cancer radiotherapy Hongxing Liu, Weiqiang Lin, Lizhen He, Tianfeng Chen PII:

S0142-9612(19)30644-1

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119545

Reference:

JBMT 119545

To appear in:

Biomaterials

Received Date: 13 September 2019 Revised Date:

12 October 2019

Accepted Date: 13 October 2019

Please cite this article as: Liu H, Lin W, He L, Chen T, Radiosensitive core/satellite ternary heteronanostructure for multimodal imaging-guided synergistic cancer radiotherapy, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119545. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Radiosensitive Core/Satellite Ternary Heteronanostructure for Multimodal Imaging-Guided Synergistic Cancer Radiotherapy Hongxing Liu, Weiqiang Lin, Lizhen He*, Tianfeng Chen* The First Affiliated Hospital, and Department of Chemistry, Jinan University, Guangzhou 510632, China. *Correspondence E-mails:[email protected] (L. H.), [email protected] (T. C.) ABSTRACT: Developing safe, effective and targeting radiosensitizers with clear action mechanisms to achieve synergistic localized cancer treatment is an important strategy for radiotherapy. Herein, we design and synthesize a ternary heteronanostructure radiosensitizer (SeAuFe-EpC) with core/satellite morphology by a simple method to realize multimodal imaging-guided cancer radiotherapy. The mechanistic studies reveal that Se incorporation could drastically improve the electrical conductivity and lower the energy barrier between the three components, resulting in more electrons transfer between Se-Au interface and migration over the heterogeneous junction of Au-Fe3O4 NPs interface. This synergistic interaction enhanced the energy transfer and facilitated more excited excitons generated by SeAuFe-EpC NPs, thus promoting the transformation of 3O2 to 1O2 via resonance energy transfer, finally resulting in irreversible cancer cell apoptosis. Additionally, based on the X-ray attenuation ability and high NIR absorption of AuNPs and the superparamagnetism of Fe3O4, in vivo computer tomography, photoacoustic and magnetic resonance tri-modal imaging have been employed to visualize the tracking and targeting ability of the NPs. As expected, the NPs specifically accumulated in orthotopic breast tumor area and achieved synergistic anticancer efficacy, but showed no toxic side effects on main organs. Collectively, this study sheds light on the potential roles of core/satellite heteronanostructure in imaging-guided cancer radiotherapy. Keyword: Radiosensitization; multimodal imaging; selenium; radiotherapy.

1. Introduction Radiotherapy (RT) is one of the most feasible noninvasive cancer therapeutic modalities and extensively applied in different clinical cancer therapy, including nasopharynx cancer, breast cancer, melanoma and mediastinal tumors[1]. However, many critical factors of solid tumors limited its therapeutic effect[2]. For instance, hypoxia[3, 4], acidic pH[5] and high-level H2O2[6]/GSH[7] in tumor microenvironment (TME) could promote tumor aggressiveness, metastasis, recurrence and resistance to RT[8, 9]. In addition, reducing the dose of radiation or increasing the times of radiation would cause radiotherapy tolerance and result in poor therapeutic efficiency or even failure of RT[10]. To overcome these limitations, numerous radiosensitizers have been widely applied in the clinical treatments to enhance the RT efficacy, such as platinum complexes, sodium glycididazole and sanazole[11-13]. However, severe side effects such as nephrotoxicity and neurotoxicity generated by these radiosensitizers limit its clinical use because of serious side effects due to its poor specificity to tumor cells[14]. Therefore, it is urgent to develop a high effective and low toxicity radiosensitizer to improve the therapeutic effect of RT. Nanomaterials demonstrate great potential as radiosensitizers due to their advantages in biomedical applications [15-18]. A variety of nanomaterials have been widely applied for enhancement of radiotherapy attributed to their versatile physicochemical properties, such as high Z elements [19-21] (including gold (Au)-[22], gadolinium (Gd)- [23], bismuth (Bi)- [24-27], and tungsten (W)-[28]based nanomaterials), semiconducting nanomaterials[29, 30] and iron-based nanomaterials[31]. Recently, studies also found that, black phosphorus[32-34] and selenium nanoparticles (SeNPs)[35] exhibited radiosensitization effects. Se is an important trace element necessary for humans and animals[36, 37]. Previously, we found that SeNPs [38] and selenadiazole derivatives [39, 40] could significantly enhance X-ray-based cancer radiotherapy by producing Compton effect and photoelectric effect to induce cell apoptosis[41]. However, the imaging ability of

Se-containing radiosensitizer is often limited [42-46], what’s more, the development process of tumor is usually intricate, and the pathogenesis of the tumor, clinical symptoms and individual differences of patients and other factors are also very sophisticated [46-48]. Therefore, it is of great significance to develop safe, highly effective and cancer-targeting radiosensitizers with multimodal imaging capability to achieve synergistic therapeutic effects and precisely tracking and guiding the therapy. Among the nanoradiosensitizers, AuNPs as high-Z and extremely high chemical inertness material had been widely used not only as radiosensitizers to enhance RT efficacy due to their large photoelectric absorption coefficient at the tumor site, but also as contrast agents for X-ray-Computed tomography Imaging (CT) and photoacoustic imaging (PA) due to its intrinsic X-ray attenuation ability and high NIR absorption[49-51]. In contrast, MRI is usually considered to be a safe diagnosis method with higher soft tissue resolution and better display of lesions than CT and without radiation damage. Therefore, it is necessary to develop a kind of radiotherapy-sensitized nanoparticles with both MRI and CT imaging ability, which can be used in multimodal imaging of tumors, and then accurately diagnose tumors by comparing the imaging results[52-54]. Superparamagnetic iron oxide nanoparticles (Fe3O4) are widely considered to be an ideal contrast agent for MRI because of its good superparamagnetism[55-58] with high biocompatibility and low toxicity to healthy tissues[59]. In addition, Fe3O4 also exhibited radiosensitization effects by catalyzing ROS formation through Fenton and Hubble-Weiss reaction [31, 60-62]. Although different types of nanomaterials have been found to show radiosensitization effects, little information is available on the chemical nature and action mechanisms accounting for this effect. How to design effective nanomedicines with multi-model imaging capability to achieve better radiotherapeutic efficacy is still the bottleneck for successful cancer therapy. Herein, we rationally design and synthesize a ternary nano-radiosensitizer (SeAuFe-EpC) with uniform core/satellite morphology by a facile method to realize multimodal imaging-guided cancer radiotherapy. This NPs could be excited by X-ray to enhance energy transfer, leading to overproduction of singlet oxygen and localized ROS outburst, resulting in

irreversible cancer cell apoptosis. Additionally, CT, PA and MRI trimodal imaging have also been employed to visualize the tracking and targeting ability of the NPs. As expected, this nano-radiosensitizer could specifically accumulate in orthotopic breast tumor area and achieve synergistic anticancer efficacy, but show no toxic side effects on the main organs. Taken together, this study demonstrates a successful strategy for cancer inhibition by using Se-functionalized nanomedicine-potentiated radiotherapy.

Scheme 1. Schematic illustration of the radioensitization mechanisms of SeAuFe-EpC NPs in multimodal imaging-guided cancer radiotherapy.

2. Materials and Methods 2.1 Materials Sodium selenite (Na2SeO3), chitosan (CS), L-ascorbic acid (Vc) were purchased from Sigma-Aldrich. Chloroauric acid hydrated (HAuCl4·4H2O), FeCl3·6H2O was obtained from Macklin Inc. (Shanghai, China) and used as received. Polyethylene glycol 1500 (MW=1500) were purchased from Aladdin Chemical Co. Ltd, China. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco®ThermoFisher Scientific Inc. The mice used in this study were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. and treated with care in compliance following the guidelines of the Care and Use of Laboratory Animals. All animal experiments were conducted under the approval of the Animal

Experimentation Ethics Committee of Jinan University. 2.2 Fabrication of SeAuFe-EpC: Fe3O4 NPs were synthesized by a solvothermal reaction as reported previously[63]. Then, 20 mg as-prepared Fe3O4 NPs were protonated with HCl aqueous solution for 5 min. Then the protonated Fe3O4 NPs were resuspend in 20 mL L-cysteine aqueous solution to obtain L-cysteine-functionalized Fe3O4 NPs. The AuFe NPs were synthesized as reported previously[64]. Then, certain concentration of Na2SeO3 was added gradually to 5 mL AuFe NPs solution and four times concentration of vitamin C added dropwisely. After 15 minutes, 0.6 mL of CS-EpCAM, which was synthesized by mixed with 2 mL of EpCAM and CS (0.8 mg/mL) under 10 µg/mL NHS and EDC activating, were added to a Milli-Q water and the final volume was set to 10 mL and mechanical stirring to react overnight. Afterwards, magnetic separation was used to remove unreacted materials. The as-prepared nanosystem was marked as SeAuFe-EpC. 2.3 Fabrication of Se-Fe3O4 NPs Before Se-Fe3O4 NPs synthesized, ~10 nm Fe3O4 NPs were synthesized by microwave synthesis. Briefly, 123.6 mg (0.35 mmol) iron acetylacetone was dissolved in 4.5 ml anhydrous benzyl alcohol, reaction at 60 °C for 5 min, 210 °C for 5 min and 60 °C for 5 min, the products were wash with 30 ml ethanol, 6000 rpm centrifugal for 20min (twice). Se-Fe3O4 NPs were synthesized according to the method reported by Xia’ group[65] by regulate the temperature to control the encapsulation of iron oxide nanoparticles and the growth of amorphous selenium (a-Se) colloids. The slow reduction rate at this temperature greatly promoted the role of iron oxide particles as exotic nuclei. By carefully temperature control, the a-Se colloids growth and the Se-Fe3O4 NPs was synthesized. 2.4 Characterization of the SeAuFe-EpC. Transmission electron microscopy (TEM), energy-dispersive X-ray spectrometry (EDS), were carried out on the JEM-2100F TEM at an acceleration voltage of 200 kV. Scanning electron microscope (SEM) was carried out on the HitachiSU8000SEM. Powder X-ray diffraction (XRD) patterns of the bulk samples were measured on an UltimaIV X-ray Diffractometer.

2.5 Magnetic properties of SeAuFe-EpC: VSM analysis was performed on PPMS DynaCool system(MPMS XL-5, 9.0T).The magnetic properties of SeAuFe-EpC solutions ranging from 0.0025-0.04mM (equivalent Fe concentration) were determined by using a 1.5 T Signa HDxt superconductor clinical MR system (GE Medical, Milwaukee, WI). The relaxivity value (r2) of SeAuFe-EpC NPs, which is defined as 1/T2, was measured with the parameters as previously described [57]. 2.6 Cell culture and MTT assays. The human breast carcinoma MCF-7 cells and Hs578BsT cell were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA). The cell viability with and without X-ray irradiation was determined by MTT as previous reported method [43]. Briefly, the cells were treated with Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs for 72 h at the equivalent concentrations of 10 µg/mL Fe. After 12 h, MCF-7 cells were irradiated with 4 Gy X-ray. After another 72 h incubation, the cytotoxicities of the combined treatment of Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs, X-ray against MCF-7 cells were measured by MTT assay. 2.7 Cell uptake analysis. The cell uptake after SeAuFe and SeAuFe-EpC NPs treatment were analyzed by flow cytometric analysis as previously reported[35]. 2.8 Flow cytometric analysis: Flow cytometry was used to quantitatively analyze the effect of SeAuFe-EpC NPs on tumor cell cycle and apoptosis ratio before and after radiotherapy. Firstly, the cells in logarithmic growth phase were inoculated in 6 cm petridish at a certain concentration (6 mL), and the cells adhered to the wall for 24 h. Then 10 µg/mL of SeAuFe-EpC nanoparticles were added and incubated for 24 h. After incubation, the cells were collected. It was fixed overnight in a refrigerator at -20 °C with precooled 70 % ethanol. The next day, ethanol was removed, 500 µL PI solutions was added to avoid light and dyed at room temperature for 2 h. These cell samples were then detected by Beckman flow cytometry, of which at least 10000 cells were detected in each sample. Finally, the cycle analysis of intracellular DNA content was carried out by Flowjo software, and the ratios of G0/G1, S, G2/M and apoptosis peak Sub G1 were obtained. 2.9 Mitochondrial morphology analysis: Mitochondrial fragmentation caused by

SeAuFe-EpC NPs in MCF-7 cells was visualized using a Mitotracker & Hoechst staining assay according to previous reported method[24]. 2.10 Characterization of transient absorption (TA) of NPs: The TA spectrum of Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs that irradiated by X-ray with dose of 4 Gy were performed on a femtosecond transient absorption system (Helios, Ultrafast, USA). 2.11 ESR measurements of SeAuFe-EpC NPs: After Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs was exposed to X-ray at 4 Gy, 1 g/L of ethanol suspension sample was mixed with the spin-trapping agent (2,2,6,6-tetramethylpiperidine, TEMP) aqueous solution. The mixture was characterized using a BRUKE A300 spectrometer operating at X-band frequency (3.5 GHz) at room temperature. 2.12 Measurement of superoxide anions. The intracellular superoxide anions generation induced by the nanosystems and X-ray was measured using DHE probe according to previous method.[48] 2.13 MRI of SeAuFe-EpC in vivo: The biodistribution of SeAuFe-EpC NPs in MCF-7-bearing orthotopic nude mice was performed in a 1.5 T MR scanner. The nude mice were divided to 4 groups randomly (n = 3 per group). 10 µg/kg Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs were intravenously injected. MRI analysis were performed by collecting the T2-weighted signal of Fe3O4 in the mice. The concentration of Fe3O4 accumulated in tumor was measured as the percentage difference of transverse relaxation rate (∆R2*). 2.14 In vivo photoacoustic imaging (PA): Balb/c nude mice with tumor size of 7~8 mm were selected and injected with SeAuFe (10 µg/mL, 0.1 mL) and SeAuFe-EpC (10 µg/mL, 0.1 mL) via intravenous injection for 8 h. The mice were anesthetized with 30 µL 2 % pentobarbital sodium and kept their body temperature constant at 37.5 °C. Photoacoustic imaging tomography scanner (Vevo 2100 LAZR) setup frequency: 40 MHz; 2D gain: 0 dB; PA gain: 28 dB; When the excitation wavelength was 680 nm, 708 nm and 808 nm, the photoacoustic signals of SeAuFe-EpC NPs were collected in turn. 2.15 In vivo computed tomography imaging (CT): Three nude mice in each group

of Balb/c were taken. The mice were anesthetized with pentobarbital sodium (2 %, wt/vol, 50 µL) after intravenous injection of Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs (10 µg/mL, 0.1 mL), at different time points (2, 4, 6, 8, 12, 24 h), and the mice were anesthetized with pentobarbital sodium (2 %, wt/vol, 50 µL). To assess the CT contrast efficacy, SeAuFe-EpC and Iopromide injection (Ultravist) at an equivalent dose of 10 mM Au and iodine were intravenously injected for CT imaging after 8-h injection, respectively. The CT scanner adopts CT X-ray tomography system (TOSHIBA, AQUILION/64), and the scanning parameters are set as follows: scanning voltage: 120 kV, current: 120 mA; FOV: 250; matrix. 512 × 512, scanning layer thickness 1 mm, layer spacing 1 mm, window width 80 HU, and window level 40 HU. 2.16 Administration mice mode and anticancer ability of SeAuFe-EpC in vivo: The MCF-7 mouse orthotopic breast cancer model was constructed by injecting 2×106 MCF-7 cells into the breast pad near the left lower extremity of BALB/c nude mice. When the tumor grow up to 150 mm3 after inoculations, the tumor-bearing mice were used for further study. The orthotopic breast cancer nude mice were randomly divided into 10 groups (n = 6 per group). Equivalent concentration of Fe3O4, AuFe, SeAuFe and SeAuFe-EpC (10 µg/kg), were intravenously injected into the mice per 2 days. After 8 h of administration, the tumor was irradiated with X-ray (4 Gy). The tumor volume and body weight of each mouse was weighted per 2 days and calculated as previous reported method[57]. The mice were sacrificed to collect blood for blood biochemical analysis after 21days of drug administration. The heart, liver, spleen, lung, kidney and tumor were exploited and weighted. 2.17 MRI acquisition: T2-weighted MRI of the nude mice with MCF-7 mouse orthotopic breast cancer models were conducted after 21 days different treatments with a 1.5 T MR scanner using a mouse body volume coil. The indexes of standard ADC, slow ADC were employed to evaluate the necrotic degree of tumor in vivo. 2.18 Hematological and histological analyses: At the end of the experiment, blood samples were collected from eyeballs, and about 500 µL serums was obtained by centrifugation, which was sent to the blood testing center of the first affiliated hospital

of Jinan University for Blood Biochemical Indexes Analysis. The analysis indexes included glutamic pyruvic transaminase (ALT); lipid related low density lipoprotein (LDLC); globulin (GLB); high-density lipoprotein (HDL-C); blood glucose (GLU); renal function related urea nitrogen (BUN) and so on. The heart, liver, spleen, lung, kidney and tumor were prepared as previous reported method[35] for histology observation and immunohistochemical analysis under an microscope. 2.19 Pharmacokinetics analysis of SeAuFe and SeAuFe-EpC in vivo: The pharmacokinetics analysis was performed as previous reported method[57]. Briefly, six female SD rats were divided into 2 groups randomly. SeAuFe and SeAuFe-EpC NPs were injected intravenously at an equivalent dosage of 10 mg Se per kg. At 1, 2, 4, 8, 12, 24, 48 and 72 h, blood was collected from the canthus of the rat eyes and centrifuge at 3000 rpm to collect the plasma from the supernatant. 200 µL plasma was digested with chloroazotic acid and set volume to 10 mL. The sample was then applied to ICP-MS (NexION 350, Perkinelmer, USA) to analyze the plasma Se concentration. 2.20 Statistics analysis: All experiments were carried out in triplicate (n=3) and results were expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 7. Difference between two groups was analyzed by two-tailed Student's-t test. Difference of multiple groups was analyzed by one unpaired multiple t test. Difference with P< 0.05 (*), P<0.01(**) or P<0.001(***) was considered statistically significant.

3. Results and Discussion 3.1 Rational design and characterization of SeAuFe-EpC NPs The ternary radiosensitizer was designed and synthesized by the sequential assembling of SeNPs, AuNPs and the target peptide (epithelial cell adhesion molecule, EpCAM) on the surface of Fe3O4. As illustrated in Scheme 1, we firstly coated AuNPs onto Fe3O4 to form a core/satellite structure. And then, SeNPs was bound to the AuNPs. After modification with EpCAM that has been reported to be able to

recognize receptors overexpressed in breast cancer [66, 67], the obtained SeAuFe-EpC NPs may exhibit excellent specific recognition of tumor cells. The size and shape of the products were investigated by SEM and TEM. The TEM images of the as-prepared Fe3O4 NPs (Figure S1) showed that the size of the Fe3O4 NPs was at about 210 nm. The representative images in Figure 1A-C showed that the morphology of SeAuFe-EpC NPs were spherical in shape with the average size at about 240 nm. The high-resolution TEM (HR-TEM) image (insert in Figure 1C) clearly shows a lattice spacing of 0.22 nm and 0.25 nm corresponding to the (111) and (331) plane of Au and Fe3O4 with some lattice mismatch observed, indicating that Au NPs is heterojuncted with Fe3O4 NPs, but not physical mix. The EDS analysis results in Figure 1D showed that Au element and Se element were observed on the surface of the Fe3O4, which further demonstrated the coating of Au and Se on Fe3O4, more interesting, the Se and Au were co-located in the same position. To investigate the way the heterostructure nanoparticles formed, X-ray photoelectron spectroscopy (XPS) analysis was further performed. The results (Figure 1E-H) showed that the characteristic peaks of Au and Se shifted slightly (~0.3 eV) because of the interaction between Au and Se, indicating that the Se is covalently bound to the AuNPs through Au-Se coordination. X-ray diffraction (XRD) was also performed to further study crystal structure. As shown in XRD spectra in Figure 1I, they agree well with the JCPDS No. 99-0056 of the Au element and JCPDS No. 99-0073 of the Fe3O4 crystal, whereas there was no characteristic peaks of Se appeared in the XRD spectra, which may be due to a low content of Se in the NPs out of the detection limit of XRD. The magnetic properties of the Magnetic microspheres were investigated with a vibrating sample magnetometer (VSM). Figure 1J showed the magnetization curves measured at 300 K and the magnetic saturation values were 75.8 emu/g for Fe3O4, 45.9 emu/g for AuFe and 30.8 emu/g for SeAuFe NPs, which confirmed that the as-prepared SeAuFe NPs is superparamagnetism and thus can be used as T2-weighted contrast agent for clinical MRI. The results of UV-vis spectra (Figure 1K) demonstrate the presence of Au and Se in the SeAuFe-EpC NPs, as evidenced by their characteristic absorbance red shift to 700 nm from a broad-spectrum absorption between 500

nm~1000 nm, which is not observed in the absorption spectra of Au NPs and Se NPs (Figure S2). The results of Fourier transform infrared (FT-IR) spectroscopy (Figure 1L) further demonstrated the peaks at 1647 cm-1 and 1563 cm-1 were assigned to the amide bonds I and II, respectively, indicating successful conjugation between CS and EpCAM. The size distribution and stability of SeAuFe-EpC NPs in PBS, DMEM with 10 % FBS and human serum were also investigated. The results (Figure S3) show that, the size of SeAuFe-EpC NPs slightly increased in DMEM (10 % FBS) comparing with PBS. Moreover, the size of the NPs remained stable in human serum up to 72 h, indicating the high stability of SeAuFe-EpC NPs under physiological conditions. These results indicate the successfully synthesis of SeAuFe-EpC NPs.

Figure 1. Synthesis and characterization of SeAuFe-EpC NPs. A) SEM micrographs of the SeAuFe-EpC NPs; B-C) The TEM images of SeAuFe-EpC NPs; insert: high-resolution TEM images of the SeAuFe-EpC NPs D) STEM-EDS elemental mapping images of the SeAuFe-EpC NPs showing the successful conjugation of AuNPs and SeNPs on Fe3O4; E) XPS spectra of Fe3O4, AuFe and SeAuFe NPs; F-H) XPS spectra of Fe2p, Au4f and Se3d; I) XRD analysis of Fe3O4, AuFe and SeAuFe NPs; J) M-H curves of Fe3O4, AuFe and SeAuFe NPs at 300 K

(insert show the excellent magnetic response capacity of the as-prepared SeAuFe-EpC NPs); K) UV absorption spectra of Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs; L) FT-IR spectra of SeAuFe-EpC NPs (a) and SeAuFe (b). 3.2 Radiosensitization ability of the ternary radiosensitizer in vitro To investigate the cytotoxicity in vitro, the cell growth inhibition effects of Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs on MCF-7 breast cancer cells and Hs578BsT human normal mammary fibroblasts were determined by MTT assay. The results (Figure 2A) showed that after treatment with Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs for 4 h, exposure to X-ray radiation (4 Gy) and cultured for another 72 hours, MCF-7 cells viability decreased significantly compared to the control. However, after treatment for 72 h, different NPs groups and X-ray alone group showed no significant inhibition on MCF-7 and Hs578BsT cell growth (Figure S4). Moreover, the combined treatment with the SeAuFe-EpC NPs and X-ray radiation inhibited growth of MCF-7 cells in a dose-dependent manner under 2 Gy, 4 Gy and 8 Gy irradiation (Figure 2B). In contrast, the combined treatment showed lower cytotoxicity against Hs578BsT normal cells (Figure S5), confirming the good selectivity of the SeAuFe-EpC NPs between cancer and normal cells. The growth of the MCF-7 cells was more significantly inhibited when co-treated with SeAuFe-EpC NPs and X-ray than single therapeutic regimens. In order to survey the role of Se in radiosensitization, Fe3O4-Se NPs were synthesized and used as control group. The results of TEM and XPS analysis (Figure S6) showed that Se NPs was just physically mixed with Fe3O4 without coordination interaction, since the Fe2p and Se3d XPS spectra of Se-Fe3O4 NPs didn’t change comparing with those of Fe3O4 NPs and Se NPs. The results of the radiosensitization ability (Figure S7) also show that Fe3O4-Se NPs demonstrated lower radiosensitization effect than AuFe NPs, SeAuFe NPs and SeAuFe-EpC NPs. The results highlight the role of Se in improving the radiosensitization effect of this nanosystem. The cellular uptake of coumarin-6-labelled SeAuFe and SeAuFe-EpC NPs in MCF-7 cells were analyzed by flow cytometry, and the results (Figure S8) indicated that SeAuFe-EpC NPs possessed higher and faster cellular uptake compared to

SeAuFe NPs in a dose-dependent manner, suggesting that SeAuFe-EpC NPs would accumulate in tumor cells more efficiently due to the EpCAM peptide modified on the nanoparticles. Then, the cellular uptake of coumarin-6-labelled SeAuFe-EpC NPs in MCF-7 cells at different times was further evaluated by flow cytometry. The results in Figure 2C showed that cellular uptake of the SeAuFe-EpC NPs was in a time-dependent manner. It appeared a significant difference in fluorescence within 1 h compared with 0 h and reached saturation within 4 h. To visually monitor the intra-cellular localization of coumarin-6-labelled SeAuFe-EpC in MCF-7 cells, lysosome marker LysoTracker (red) and nucleus marker Hoechst 33342 was used to stain the nucleus and mitochondria, respectively. As illustrated in Figure S9, green fluorescence appeared in the cell membrane after 1-h incubation while a superimposed color can be found between the green and the red fluorescence after 4 h, which was in accordance with the results of cellular uptake by flow cytometry. These results suggest that lysosome is the main target organelle of the SeAuFe-EpC NPs. TEM images of ultrathin cell slices was used to directly examine ultrastructure localization and morphology of SeAuFe-EpC NPs in MCF-7 cells. As shown in Figure 2D, after treatment with 10 µg/mL SeAuFe-EpC NPs for 4 h, SeAuFe-EpC NPs were obviously found in the intracellular cellular vesicles of MCF-7 cells. The enlarged images showed that some broken SeAuFe-EpC NPs could be found in the intracellular vesicles. These results demonstrate that SeAuFe-EpC NPs could indeed be internalized by the tumor cells. Furthermore, we conducted the clonogenic assay to confirm the radiosensitization effect of the nanoparticles. As shown in Figure 2E-F, the number of colonies in the control group and Fe3O4, AuFe, SeAuFe, and SeAuFe-EpC NPs group was the largest, indicating that the different components of the ternary radiotherapy sensitizer didn’t affect cell proliferation and exhibited outstanding biocompatibility. While the X-ray group had a slightly lower number of cell colonies, which was consistent with the clinical use of X-ray RT on tumors. In this regard, to verify whether each constitute of the ternary naosensitizer is able to significantly inhibit the cell colony formation or not. The Fe3O4, AuFe, SeAuFe, and

SeAuFe-EpC NPs were irradiated with 4 Gy of X-rays, respectively. The results showed that the survival fraction of each co-treated group decreased from 52 % to 13 %. The cloning experiment showed that cancer cell proliferation was significantly inhibited by this radiosensitizer. Studies were also carried out to examine the radiosensitization effect of the SeAuFe-EpC NPs on cell cycle progression. As revealed by flow cytometris analysis in Figure 2G and Figure S10, X-rays alone caused 7.96 % Sub-G1 apoptosis of MCF-7 cells and the Fe3O4, AuFe, SeAuFe, and SeAuFe-EpC NPs increased the cell death from 2.08 % to 10.21%, respectively, while co-treatment increased it from 9.12 % to 29.92 %. The results indicate that the individual component of the ternary radiosensitizer is almost non-toxic, while under X-ray irradiation, the different components of the ternary radiosensitizer can significant enhance the effect of the radiation in vitro. Mitochondria are essential in different cellular events. However, a lot of factors may injure the morphology and functions of mitochondria, and lead to further cell apoptosis and cell death[68]. Therefore, the mitochondrial fragmentation was assessed to verify mitochondrial dysfunction induced by SeAuFe-EpC NPs combined with radiotherapy. As shown in Figure 2H, the mitochondria and nucleus were stained with MitoTracker Red and Hoechst 33342 respectively. The typical photos of mitochondria showed that the mitochondria exhibited red thread-like filaments morphology in control group, while the combined SeAuFe-EpC NPs with radiotherapy treatment group generated seriously mitochondrial fragmentation. It should be note that the SeAuFe-EpC NPs or X-rays alone did almost no damage to the cell's mitochondria. These results suggest that the combined treatment could more potently cause mitochondria dysfunction.

Figure 2. Radiosensitization effect of SeAuFe-EpC NPs in vitro A) Cytotoxic effects of different NPs at 10 µg/mL with or without X-rays on MCF-7 cells for 72 h. B) Cell viability of MCF-7 cells treated at different doses (0, 2, 4, 8 Gy) of X-rays for 72 h incubation with 10 µg/mL SeAuFe-EpC NPs; C) Cell uptake analysis results of the SeAuFe-EpC NPs at different times; D) TEM examination of ultrathin MCF-7 cell slices treated with 10 µg/mL SeAuFe-EpC NPs for 4 h; E) Fe3O4, AuFe, SeAuFe and SeAuFe-EpC NPs enhance the inhibitory effects of X-rays on the colony formation of MCF-7 cells; F) Cloning analysis of different NPs on MCF-7 cells after different dose of radiation; G) Flow cytometric analysis of different NPs on cell cycles with or without of X-rays; H) The morphological change of mitochondria in MCF-7 cells treated with SeAuFe-EpC NPs and X-rays (4 Gy), scale bar = 100 µm.

3.3 Radiosensitization mechanisms of SeAuFe-EpC NPs In order to further analyze the radiosensitization mechanism of SeAuFe-EpC NPs, the fs-resolved transient absorption (TA) spectrum technique, which is usually used to analyze the real-time ultrafast relaxation kinetics of tested materials, was employed to examine the ultrafast photoinduced carrier dynamics of the SeAuFe-EpC NPs[69, 70]. In this study, the NPs with or without radiotherapy was excited with one pump light (wavelength: 400 nm) and probed with another variable delay later (1 kHz, 400 nm-720 nm). By measuring the wavelength-dependent absorption difference of the probe light (∆A), the influence of X-ray on the sample can be determined. Two life time can be extracted from the decay curve of sample by a double exponential decay function fitting analysis, expressed asy = ‫ݕ‬଴ + ‫ܣ‬ଵ ݁ ି(௫ି௫బ )ఛభ + ‫ܣ‬ଶ ݁ ି(௫ି௫బ )ఛమ . As shown in Figure 3A, the fast decay is attributed to the photoexcited electron relaxation from S1 state to T1 state (τ1), while the slow decay process is related to nonradiative recombination of photoexcited carriers (τ2). Figure 3B showed that the slow decay time τ2 of SeAuFe-EpC NPs became shorter after radiotherapy from 242.76 ps to 219.59 ps. Figure 3C and Figure S11 shows the TA spectra (600-720 nm) of different nanoparticles with or without X-ray (4 Gy) irradiation taken at several representative probe delays (1.14, 4.43, 8.04, 32 and 106 ps). More importantly, after radiation, τ2 time was significantly reduced from 321.93 ps to 242.14 ps after coating Se onto Au NPs. It is possible that Se incorporation can drastically improve the electrical conductivity and lower the energy barrier among these three components, resulting in more electrons transfer between Se and Au interface[71]. The results show the similar spectral profiles change presented as photoinduced absorption and the slow decay process is assigned to the strong many-body effects, which lead to efficient generation of excited excitons between different components of the SeAuFe-EpC NPs, thus promotes the transformation of oxygen atom at triplet state to singlet oxygen via resonance energy transfer[72]. We inferred that the decreased lifetime in X-ray-excited sample, was accounted to the enhanced energy transfer efficiency after X-ray excitation. This gives rise to the enhanced Compton-, Photo-, or Auger-electrons and then induced the secondary electron. Those secondary electrons

not only induce ROS production but also migrate over the Au-Fe3O4 interface to the Fe3O4 NPs due to the heterojunction structure of Au-Fe3O4 NPs, resulting in more ROS production on the Fe3O4 NPs surface[26, 50]. It is the synergistic interaction among these three components contributes together to the enhanced energy transfer, which facilitates more excited excitons generated between different components of SeAuFe-EpC NPs. As a result, the ROS generation efficiency was dramatically enhanced by the excitation of high photon energy of X-ray. Therefore, the shorter slow decay time of SeAuFe-EpC contributes to more effective energy transfer, and thus causes higher level of ROS generation. In order to further verify our hypothesis, electron spin resonance (ESR) spectroscopy (Figure 3D) was used to identify the ability of different components of SeAuFe-EpC NPs to produce singlet oxygen with the assistance of 2, 2, 6, 6-tetramethylpiperidine (TEMP), a typical capture-agent for short-lived singlet oxygen. It was found that only after X-ray radiate to the Fe3O4, AuFe, SeAuFe, and SeAuFe-EpC NPs that three characteristic signals in the ESR spectrum could be seen. It should be noted that the spectral signal was gradually enhanced as the SeNPs and AuNPs sequentially coated onto Fe3O4 compared with control which no nanoparticles were added. The results suggested that X-ray-excited sample indeed promoted more singlet oxygen production. The overproduction of superoxide anions induced by radiotherapy was also examined by the DHE probe. As shown in Figure 3E-F, the Fe3O4, AuFe, SeAuFe, and SeAuFe-EpC NPs-treated group increased the superoxide anions level slightly, while the content of superoxide anions increased significantly in the SeAuFe-EpC NPs combine with X-ray irradiation group. As shown in Figure 3E, SeAuFe-EpC NPs alone increased the superoxide anions level to 130% within 20 min, while the combined application of SeAuFe-EpC NPs and X-ray treatment increased the superoxide anions content to 225% within 20 min and remained stable due to persistent cellular uptake of SeAuFe-EpC NPs. The results indicate that the SeAuFe-EpC NPs and X-ray elevated singlet oxygen and superoxide anions generation synergistically and lead to the tumor apoptosis.

Figure 3. Radiosensitization mechanism of SeAuFe-EpC NPs. A) Schematic diagram of ROS generated on nanoparticles after radiotherapy; B) Transient absorption (TA) spectrum analysis of spectral lifetime changes before and after radiotherapy; C) TA spectra (600-720 nm) of SeAuFe-EpC NPs with or without X-ray (4 Gy) irradiation taken at several representative probe delays (1.14, 4.43, 8.04, 32 and 106 ps); D) Electron spin resonance (ESR) spectroscopy analysis the ability of different nanoparticles to produce singlet oxygen; E) DHE probe to detect the amount of superoxide anions produced by different nanoparticles in cells; 1： SeAuFe-EpC+X-Ray，2：SeAuFe+X-Ray，3：AuFe+X-Ray，4：Fe3O4+X-Ray， 5：SeAuFe-EpC，6：SeAuFe，7：AuFe，8：Fe3O4. F) Representative photos of superoxide anions produced by different nanoparticles. 3.4 Imaging-guided therapy in vivo Firstly, different concentrations of SeAuFe-EpC NPs were applied for MR imaging analysis in vitro, and the results (Figure 4A) demonstrated the dose-dependent property of the MR signal of this NPs, with r2 value found at 69.8 mM-1 s-1 by the linear relationship of the longitudinal relaxation rate (1/T2) vs. Fe concentration, suggesting the MR imaging capacity of the SeAuFe-EpC NPs. Therefore, the accumulation of SeAuFe-EpC NPs in tumor region was monitored by capturing the T2-weighted signal of SeAuFe-EpC NPs under a 1.5 T MR scanner. The in vivo results showed a stronger accumulation of SeAuFe-EpC NPs in tumor region in 48 h than that of Fe3O4, AuFe or SeAuFe NPs treatment, as presented by the stronger T2-weighted signal (Figure 4B) and the percentage difference of transverse

relaxation rate △R2* of SeAuFe-EpC NPs maximized at 8 hours (Figure 4C). Prussian blue staining was also use to detect the Fe content in tumor observed in a period of 48 h to prove tumoral accumulation of SeAuFe-EpC and the results (Figure S12) show that no positive staining was found in the control group at 0 h, while, some scattered blue dots appeared at 4 h. The characteristic blue dots reached the highest level and dropped to a lower level at 12 h but keep it constant in next 36 h due to the EPR effect of the tumor and the target peptide modified on the nanoparticles. These results are consistent with those of MRI imaging. What’s more, the values of CT signal were proportional to the concentration of SeAuFe-EpC NPs (R2 = 0.99) in Figure 4D. We next conducted the CT imaging of the NPs on MCF-7 orthotopic breast tumor mice. It was found that strong CT imaging signals in the tumor area could be found after 8 h injection compared with the tumor site before the NPs injection (Figure 4E-F), due to the specific target recognition of EpCAM antigens on MCF-7 cells and the EPR effect. We also investigated the CT imaging capacity of SeAuFe-EpC NPs comparing with clinically used iodine-based CT contrast agent Iopromide (Ultravist) after i.v. injection. The results showed that SeAuFe-EpC NPs could provide an approximate CT contrast effect at equivalent concentrations comparing with Iopromide (Figure S13). These results suggest that the SeAuFe-EpC NPs could be used to guide the tumor theranostics as a highly effective CT imaging agent. Meanwhile, by comparing the results of CT with those of MRI, 8 h after administration is the better time point for cancer radiosensitization. Moreover, as shown in Figure 4G, better pronounced PA imaging signals could be observed as the concentrations of the NPs increased under 808 nm NIR irradiation (R2=0.99574), suggesting that SeAuFe-EpC NPs possess excellent in vitro PA imaging capacity. We then conducted the PA imaging of the SeAuFe-EpC NPs on MCF-7 tumor-bearing mice. As shown in Figure 4H-I, scarcely PA signals could be observed in tumor area before injection, while remarkably enhanced PA signals could be found after 680, 708, and 808 nm laser irradiation in the tumor area, respectively. In the further study, 808 nm was selected as the optimized excited wavelength based on its better tissue penetration ability and highest PA imaging signal under irradiation. The results

suggest that the SeAuFe-EpC NPs could be used as an efficient PA imaging agent at wide range of NIR wavelengths.

Figure 4. Multimode imaging analysis of SeAuFe-EpC NPs. A) In vitro MR images of SeAuFe-EpC solutions at different concentrations (Fe concentration, 0, 0.0025, 0.005, 0.01, 0.02, and 0.04 mM respectively); B) Tumoral accumulation of SeAuFe-EpC NPs observed within 48 h. The circled dotted line represented the tumor area in each nude mouse, scale bar = 5 mm; C) The percentage difference of △R2* value of tumor region from MCF-7 tumor-bearing nude mice after treated by NPs (1: Fe3O4, 2: AuFe, 3: SeAuFe, 4: SeAuFe-EpC); D) In vitro CT images of SeAuFe-EpC NPs solutions at different concentrations (0, 0.63, 1.25, 2.5, 5, and 10 µg/mL respectively; E) The 3D in vivo CT images of MCF-7 tumor-bearing mice obtained pre and post intravenous injection of SeAuFe-EpC NPs (20 µg/kg), scale bar = 20 mm; F) Statistic analysis of the CT image in vivo; G) In vitro PA images of SeAuFe-EpC solutions at different concentrations (0, 0.63, 1.25, 2.5, 5, and 10 µg/mL respectively; H) Representative photos of PA images before and after intravenous injection orthotopic breast cancer mouse model employing different NIR wavelengths (680, 780, and 808nm) (Elliptic dotted line area is collection area of photoacoustic signal); I) Statistic analysis of the PA image in vivo.

3.5 In Vivo Radiosensitization of SeAuFe-EpC NPs Mouse orthotopic breast tumor model using MCF-7 cells was established to validate the therapeutic effect of SeAuFe-EpC NPs in vivo. As shown in Figure 5A-B, representative photo of mouse tumors clearly showed the outstanding therapeutic effect of SeAuFe-EpC NPs compared with other nanoparticles. Relative tumor volume and weight changes (Figure 5C-D) indicate that the experimental group treated with X-ray by intravenous injection of SeAuFe-EpC NPs could significantly inhibited the growth of orthotopic breast tumors. No significant difference in body weight of the mice was observed in treatment and control groups within 21 days (Figure 5E), indicating that the nanoparticles did not exhibit systemic toxic side effects in nude mice. At the same time, the H&E section results of the tumor (Figure 5F) also showed that the experimental group irradiated with X-ray by intravenous injection of SeAuFe-EpC NPs significantly promoted apoptosis of tumor site cells. Immumohistochemical staining of cleaved-caspase-3 and TUNEL staining have also been performed to evaluate the apoptosis in tumor tissue. Both results presented the highest level of cell apoptosis in combined treatment group (Figure 5F and Figure S14). We further analyzed the in vivo antitumor activity of SeAuFe-EpC NPs by MRI and obtained T2-weighted MRI images from orthotopic breast tumor model after 21 days of treatment. As shown in Figure 6A, the tumor volume was significantly smaller than that of the control group after injection of SeAuFe-EpC NPs in the tail vein and X-ray treatment. As shown in Figure 6B, the number of cells in the SeAuFe-EpC NPs combine X-ray irradiated treated group was significantly reduced, indicating that the tumor was significantly necrotic compared with the other groups. While, no significant damage was observed in other major organs of the mouse. In addition, slow apparent diffusion coefficient ADC (slow ADC) value was used to examine the cell density and activity in the tumor region. As shown in Figure 6C, the slow ADC value of the X-ray group was increased by injecting SeAuFe-EpC NPs into the tail vein. What’s more, fast ADC values (fast ADC) are associated with hemoperfusion. After treatment, the fast ADC value decreased. These results

demonstrate that SeAuFe-EpC NPs could reduce the activity and density of cancer cells after radiotherapy. Taken together, these results suggest that the combination of X-rays and SeAuFe-EpC NPs significantly inhibits the growth of orthotopic breast tumor in vivo. To evaluate the potential toxicity and side effects of the SeAuFe-EpC NPs, all mice were sacrificed and their blood and major organs (heart, liver, spleen, lung and kidney) were collected for blood biochemical indexes and histopathology analysis, respectively. As shown in Figure 7A, the biochemical indicator level of ALT, LDLC, GLB, HDL-C, GLU and BUN is similar to that of healthy mice. As shown in Figure 7B, H&E sections of the main organs of the treatment group did not exhibit obvious acute toxicity of the major organs compared with the control group. The biodistribution of SeAuFe NPs and SeAuFe-EpC NPs was further assessed in orthotopic breast tumor mouse model (Figure S15), by measuring the concentration of Se and Au in different organs and tumors after 21 days of treatment, which demonstrate the effective tumoral accumulation of SeAuFe-EpC NPs than SeAuFe NPs in vivo. Possibly, the lower Se contents in the organs and tumors, could firstly be due to the lower content of Se (7.15 %) in SeAuFe-EpC NPs than Au (28.6 %). More importantly, Se could participate in the formation of selenoprotein and be easily metabolized and cleared by the mice via breath and urine [36, 73]. Interestingly, SeAuFe-EpC NPs could mainly accumulate in the liver, spleen and tumor area, which demonstrate that the SeAuFe-EpC NPs could metabolize by the liver and kidney and accumulate in tumor area for imaging-guided radiotherapy. The pharmacokinetics analysis of SeAuFe-EpC NPs (Figure S16) reveal that SeAuFe-EpC NPs drastically increased the plasma Se concentration comparing with SeAuFe NPs group, indicated by the increased indices of t1/2β (blood-elimination half-life), Cmax (maximum plasma concentration), AUC0 –72 h (area under the curve), MRT (mean retention time) and reduced Cl (clearance of medicine) value (Table S1). These results demonstrate that the targeting design of the NPs could improve the pharmacokinetics behavior of SeAuFe NPs, specific recognition of tumor antigens on MCF-7 cells and the EPR effect during blood circulation.

Figure 5. Therapeutic effect of SeAuFe-EpC NPs in vivo. A) The therapeutic effect of different nanoparticles after radiotherapy on mouse orthotopic breast tumors, scale bar = 20 mm; B) Represent photos of mouse tumors after 21 days therapy; C) Curves of relative tumor volume; D) Statistical results of relative tumor weights; E) Curves of body weights of mice, 1: PBS, 2: Fe3O4, 3: AuFe, 4: SeAuFe, 5: SeAuFe-EpC, 6: Control+X-Ray, 7: Fe3O4+X-Ray, 8: AuFe+X-Ray, 9: SeAuFe+X-Ray, 10: SeAuFe-EpC+X-Ray; F) H&E-stained, cleaved-caspase-3 and TUNEL staining tumor region from different nanoparticles with X-Ray irradiation treatment groups.

Figure 6. MRI Evaluation of in vivo antitumor activity of SeAuFe-EpC NPs. A) Typical T2-weighted MR images, fast ADC images and slow ADC images of orthotopic breast cancer mouse model after treatment with different nanoparticles (20 µg/kg) and X-ray (4 Gy) for 21 days, respectively; B) Statistic analysis of fast ADC and C) slow ADC. 1: Control, 2: Fe3O4, 3: AuFe, 4: SeAuFe, 5: SeAuFe-EpC, 6: Control+X-Ray, 7: Fe3O4+X-Ray, 8: AuFe+X-Ray, 9: SeAuFe+X-Ray, 10: SeAuFe-EpC+X-Ray.

Figure 7. In vivo toxicity evaluation: A) ALT, LDLC, GLB, HDL-C, GLU and BUN in orthotopic breast cancer mouse model after treated with different nanoparticles (20 µg/kg) and X-ray (4 Gy) for 21 days, respectively; B) H&E staining of the heart, liver, spleen, lung, and kidney before and after the treatment of different nanoparticles (20 µg/kg) and X-ray (4 Gy) for 21 days. 1: Control, 2: Fe3O4, 3: AuFe, 4: SeAuFe, 5: SeAuFe-EpC, 6: Control+X-Ray, 7: Fe3O4+X-Ray, 8: AuFe+X-Ray, 9: SeAuFe+X-Ray, 10: SeAuFe-EpC+X-Ray.

4. Conclusions In summary, we have demonstrated a ternary radiosensitizer with multimodal imaging and radiosensitive effect to achieve enhanced anticancer ability. The mechanistic study reveals that Se incorporation could drastically improve the electrical conductivity and lower the energy barrier between the three components, fascinating more electrons transfer between Se-Au interface and migration over the heterogeneous junction of Au-Fe3O4 NPs interface. The synergistic interaction among these components enhanced energy transfer and facilitated more excited excitons generated by SeAuFe-EpC NPs, thus promoting the transformation of 3O2 to 1O2 via

resonance energy transfer, finally resulting in irreversible cell apoptosis. The in vitro and in vivo experiments reveal that this nanosystem is capable of being used as CT, PA and MRI tri-modal imaging agent for precisely tracking and guiding of therapy. Therefore, in future clinical application, under the guidance of multimodal imaging, the cancer-targeted radiosensitizer can specifically accumulate in tumor area and achieve synergistic anticancer effect. Collectively, this study sheds light on the potential roles of core/satellite heteronanostructure in imaging-guided cancer radiotherapy.

Acknowledgments This work was supported by National Natural Science Foundation of China (21877049, 21701051), Major Program for Tackling Key Problems of Industrial Technology in Guangzhou (201902020013), Dedicated Fund for Promoting High-Quality Marine Economic Development in Guangdong Province (GDOE-2019-A31), and China Postdoctoral Science Foundation (2018M633273, 2016M600705).

Conflict of Interest The authors declare no conflict of interest.

Appendix A. Supplementary Data. Supplementary data to this article can be found online

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Highlights 1. A radiosensitive core/satellite ternary heteronanostructure SeAuFe-EpC was synthesized to achieve synergistic cancer radiotherapy; 2. Selenium incorporation could enhance energy transfer and facilitate more 1O2 generated; 3. The radiosensitizer could specifically accumulate in orthotopic breast tumor area but safe to the main organs; 4. The radiosensitizer enable tri-modal imaging –guided cancer radiotherapy.

Graphical abstract

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: