A Cell-Penetrating Peptide Modified Cu2−xSe/Au Nanohybrid with Enhanced Efficacy for Combined Radio-Photothermal Therapy

Radiotherapy (RT) is one of the main clinical therapeutic strategies against cancer. Currently, multiple radiosensitizers aimed at enhancing X-ray absorption in cancer tissues have been developed, while limitations still exist for their further applications, such as poor cellular uptake, hypoxia-induced radioresistance, and unavoidable damage to adjacent normal body tissues. In order to address these problems, a cell-penetrating TAT peptide (YGRKKRRQRRRC)-modified nanohybrid was constructed by doping high-Z element Au in hollow semiconductor Cu2−xSe nanoparticles for combined RT and photothermal therapy (PTT) against breast cancer. The obtained Cu2−xSe nanoparticles possessed excellent radiosensitizing properties based on their particular band structures, and high photothermal conversion efficiency beneficial for tumor ablation and promoting RT efficacy. Further doping high-Z element Au deposited more high-energy radiation for better radiosensitizing performance. Conjugation of TAT peptides outside the constructed Cu2−xSe/Au nanoparticles facilitated their cellular uptake, thus reducing overdosage-induced side effects. This prepared multifunctional nanohybrid showed powerful suppression effects towards breast cancer, both in vitro and in vivo via integrating enhanced cell penetration and uptake, and combined RT/PTT strategies.


Introduction
Cancer has been the chief reason of death worldwide. Among primary therapeutic strategies, radiotherapy (RT) is a powerful one used for over a century, which noninvasively exerts tumoricidal effects by damaging DNA directly and generating cytotoxic reactive oxygen species (ROS) indirectly [1,2]. However, X-ray irradiation alone faces multiple challenges, such as the inevitable harms to neighboring normal tissues and radioresistance induced by tumor hypoxia [3,4]. Various radiosensitizers aimed at enhancing X-ray absorption in tissues have been developed, and it is essential for them to establish the properties of stability, good biocompatibility, and tumor targeted delivery and uptake for effective clinical practice. With the development of nanotechnology, nanoparticle radiosensitizers provide versatile strategies in order to deal with these problems [5]. Among them, high-Z elements (e.g., Au [6,7], Hf [8,9], Bi [10,11], and Gd [12])-containing nanoparticles can effectively absorb high-energy radiation, and therefore can be employed as radiosensitizers to heighten the response of tumor cells to radiation. Furthermore, semiconductor nanoparticles, such as Bi 2 Se 3 [13], WS 2 [14], Bi 2 S 3 [15], and Cu 2−x Se [16], have also been extensively explored as radiosensitizers and can generate ROS based on their particular band structures. nanostructure holds the potential to act as carriers for multifunctional agents, such as chemotherapeutic drugs [33], photosensitizer [34], photothermal agents [35], and so on, further expanding its application. Thus, this work highlights an excellent multifunctional nanoplatform based on Cu2−xSe/Au-TAT nanoparticles for combined RT/PTT therapy.

Synthesis and Characterization of Cu2−xSe/Au-TAT Nanoparticles
The synthesis process of Cu2−xSe/Au-TAT nanoparticles is shown in Scheme 1A. Cu2−xSe/Au-TAT nanoparticles were synthesized via a multistep synthesis process. Briefly, in the presence of polyvinylpyrrolidone (PVP), Cu2O nanoparticles were synthesized by reducing copper chloride dihydrate (CuCl2⋅2H2O) with hydrazine hydrate (N2H4⋅H2O). Then, Cu2−xSe nanoparticles were prepared by a sacrificial template method with Cu2O nanoparticles as the sacrificial template and selenium powder as the selenium source. Afterward, Cu2−xSe/Au nanoparticles were prepared through a spontaneous redox process using hydrogen tetrachloroaurate (HAuCl4) as the source of Au. Finally, TAT was modified on the surface to improve the cellular uptake. Transmission electron microscopy (TEM) was employed to study the characteristics of size and morphology of the nanomaterials. As illustrated in Figure 1A, Cu2O nanoparticles were solid spheroids with an average size of 95 ± 5 nm, while Cu2−xSe nanoparticles maintained the spherical morphology with a hollow structure (136 ± 3 nm) ( Figure 1B). After in situ deposition of Au on the surface of Cu2−xSe nanoparticles, the average particle sizes of Cu2−xSe/Au nanoparticles (154 ± 6 nm) increased compared with Cu2−xSe nanoparticle ( Figure 1C). After surface modification with TAT, the generated Cu2−xSe/Au-TAT nanoparticles exhibited increased surface roughness ( Figure 1D). The composition of nanoparticles was further verified by elemental mapping. From Figure 1E, it is clearly displayed that Cu2−xSe/Au nanoparticles contained Cu, Se, and Au, and the Au element was evenly distributed on the Cu2−xSe nanoparticles. According to the dynamic light scattering (DLS) assessment, compared with the particle size of Cu2−xSe/Au, the size of the final Cu2−xSe/Au-TAT increased slightly to 154 ± 6 nm ( Figure 1F). As illustrated in Figure 1G, the zeta potentials of Cu2O, Cu2−xSe, and Cu2−xSe/Au nanoparticles were −9.3 ± 0.6, −18.4 ± 0.8, and −12.9 ± 0.7 mV, respectively,

Synthesis and Characterization of Cu 2−x Se/Au-TAT Nanoparticles
The synthesis process of Cu 2−x Se/Au-TAT nanoparticles is shown in Scheme 1A. Cu 2−x Se/Au-TAT nanoparticles were synthesized via a multistep synthesis process. Briefly, in the presence of polyvinylpyrrolidone (PVP), Cu 2 O nanoparticles were synthesized by reducing copper chloride dihydrate (CuCl 2 ·2H 2 O) with hydrazine hydrate (N 2 H 4 ·H 2 O). Then, Cu 2−x Se nanoparticles were prepared by a sacrificial template method with Cu 2 O nanoparticles as the sacrificial template and selenium powder as the selenium source. Afterward, Cu 2−x Se/Au nanoparticles were prepared through a spontaneous redox process using hydrogen tetrachloroaurate (HAuCl 4 ) as the source of Au. Finally, TAT was modified on the surface to improve the cellular uptake. Transmission electron microscopy (TEM) was employed to study the characteristics of size and morphology of the nanomaterials. As illustrated in Figure 1A, Cu 2 O nanoparticles were solid spheroids with an average size of 95 ± 5 nm, while Cu 2−x Se nanoparticles maintained the spherical morphology with a hollow structure (136 ± 3 nm) ( Figure 1B). After in situ deposition of Au on the surface of Cu 2−x Se nanoparticles, the average particle sizes of Cu 2−x Se/Au nanoparticles (154 ± 6 nm) increased compared with Cu 2−x Se nanoparticle ( Figure 1C). After surface modification with TAT, the generated Cu 2−x Se/Au-TAT nanoparticles exhibited increased surface roughness ( Figure 1D). The composition of nanoparticles was further verified by elemental mapping. From Figure 1E, it is clearly displayed that Cu 2−x Se/Au nanoparticles contained Cu, Se, and Au, and the Au element was evenly distributed on the Cu 2−x Se nanoparticles. According to the dynamic light scattering (DLS) assessment, compared with the particle size of Cu 2−x Se/Au, the size of the final Cu 2−x Se/Au-TAT increased slightly to 154 ± 6 nm ( Figure 1F). As illustrated in Figure 1G, the zeta potentials of Cu 2 O, Cu 2−x Se, and Cu 2−x Se/Au nanoparticles were −9.3 ± 0.6, −18.4 ± 0.8, and −12.9 ± 0.7 mV, respectively, while the zeta potential of Cu 2−x Se/Au-TAT nanoparticles was reversed to +25.1 ± 1.8 mV after surface modification with TAT. The results further clarified the successful introduction of TAT on Cu 2−x Se/Au nanoparticles. The stability of Cu 2−x Se/Au-TAT nanoparticles in aqueous solution was also detected by DLS, as demonstrated in Figure 1H; the minor variation in the hydrodynamic size in 5 days indicated Cu 2−x Se/Au-TAT nanoparticles had a good stability. UV−vis−NIR spectra was utilized to study the optical features of different nanoparticles. Figure 1I shows that the strong localized surface plasmon resonance (LSPR) of Cu 2−x Se nanomaterials was weakened after the formation of Cu 2−x Se/Au nanoparticles, and the absorption at about 560 nm that appeared in Cu 2−x Se/Au nanoparticles was attributed to the LSPR of Au nanoparticles, which further proves that Au nanoparticles were effectively deposited on Cu 2−x Se nanoparticles.
An X-ray photoelectron spectroscopy (XPS) analytical experiment was employed to study the valence states of elementary substances in Cu 2−x Se nanoparticles and Cu 2−x Se/Au nanoparticles ( Figure 1J-L and Figure S1). As shown in Figure S1C, the two strong peaks situated at 932.8 eV and 952.7 eV, accounting for 83%, corresponded to Cu 2p 3/2 and Cu 2p 1/2 of Cu (I), respectively, indicating that Cu (I) was the main valence state of Cu 2−x Se. While the two weak peaks located at 934.4 eV and 953.9 eV, accounting for 17%, corresponded to Cu 2p 3/2 and Cu 2p 1/2 of Cu (II), accordingly, demonstrating that Cu (II) also accounted for a small part of Cu 2−x Se. Moreover, the binding energy peak at 54.8 eV represents the characteristic Se 3d, corresponding to Se (II) ( Figure S1D). As shown in Figure 1J for Cu 2−x Se/Au nanoparticles, the content of Cu (I) decreased to 61% and the content of Cu (II) increased to 39% compared with the content in Cu 2−x Se nanoparticles, which proves that a part of Cu (I) was oxidized to Cu (II) during the generation process of Cu 2−x Se/Au nanoparticles. From Figure 1K and Figure S1D, it can be seen that the valence state of selenium does not change from Cu 2−x Se to Cu 2−x Se/Au. The binding energy of Au 4f 5/2 and 4f 7/2 located at 87.8 eV and 84.0 eV were attributed to Au (0), which proves that Au (III) was reduced to Au (0) ( Figure 1L). Further, the compositions of Cu 2−x Se nanoparticles and Cu 2−x Se/Au nanoparticles were determined by an inductively coupled plasma optical emission spectrometer (ICP-OES). The Cu/Se atomic ratio in Cu 2−x Se nanoparticles was 1.81:1, showing copper deficiency characteristics. The Cu/Se/Au atomic ratio in Cu 2−x Se/Au nanoparticles is 0.48:1:0.51. The reduction of Cu/Se ratio in Cu 2−x Se/Au nanoparticles further supported the reduction of Au (III) by Cu (I) in the XPS results. All of the above results clarify the spontaneous redox process from Cu 2−x Se nanoparticles to Cu 2−x Se/Au nanoparticles after the addition of HAuCl 4 solution into Cu 2−x Se nanoparticles.

Photothermal and Radiosensitizing Performances of Cu 2−x Se/Au-TAT Nanoparticles
Cu 2−x Se nanoparticles can be utilized as efficient photothermal agents thanks to the characteristic of strong LSPR in the region of NIR [16,[24][25][26]. The photothermal features of Cu 2−x Se/Au-TAT nanoparticles were researched by photothermal conversion tests. As visually illustrated in Figure 2A, after the solution of Cu 2−x Se/Au-TAT nanoparticles (80 µg/mL) being performed with a 10-min 808 nm irradiation at 0.5, 1.0, and 1.5 W/cm 2 , the temperatures of the solutions increased to 45 • C, 53 • C, and 58 • C, respectively, which indicates that the increase in temperature had positive noteworthy correlation with irradiation dose. The increase in temperature in solutions of Cu 2−x Se/Au-TAT nanoparticles also presented a concentration-dependent manner ( Figure 2B). As visually displayed in Figure 2F, the temperature of Cu 2−x Se/Au-TAT nanoparticles could increase to 53 • C under a 10-min irradiation at 1.0 W/cm 2 , while it was difficult for the water temperature to go up, showing that Cu 2−x Se/Au-TAT nanoparticles had excellent photothermal properties. According to previous literature, NIR-triggered photothermal therapy usually requires hyperthermia of >50 • C to achieve the thorough ablation of tumor [36], while extremely higher temperatures could damage normal cells. For this reason, Cu 2−x Se/Au-TAT nanoparticles at the concentration of 80 µg/mL under a 10-min laser irradiation at 1.0 W/cm 2 were chose for the conditions of evaluating the subsequent photothermal ablation effects towards cancer cells. Moreover, the photothermal stability of Cu 2−x Se/Au-TAT nanoparticles was studied by NIR laser irradiation repeatedly for 5 cycles ( Figure 2C), and the decrease in peak value of temperature with the addition of cycle was ignorable, indicating excellent photothermal stability. According to the equations listed in the Materials and Methods section, the time constant (τ) was 328.96 s ( Figure 2D,E), and the photothermal conversion efficiency (η) of Cu 2−x Se/Au-TAT was approximately 64.6%, determined by a previously reported method [37]. The photothermal conversion efficiency of Cu 2−x Se/Au-TAT is higher than some inorganic nanoparticles, such as CuO@AuCu-TPP (37.9%) [38], hollow Cu 2 Se nanozymes (50.9%) [39], and many organic nanoparticles, such as polypyrrole nanoparticles (22.6%) [40] and polydopamine-contained hydrogel (45.7%) [37]. Previous literatures had shown that the high-Z element and semiconductor heteronanostructures endow nanomaterials with better radiosensitization performance than high-Z element or semiconductor alone [26,41]. In order to verify the radiosensitization of Cu 2−x Se/Au-TAT nanoparticles, ROS generation in solution was investigated by using a probe compound 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). As can be demonstrated from Figure 2G, the fluorescence intensity of the group of Cu 2−x Se + RT was largely higher than control + RT, while the fluorescence intensities observed in the group of Cu 2−x Se/Au + RT and Cu 2−x Se/Au-TAT + RT were higher compared to those in the Cu 2−x Se+RT group, which indicated that high-Z element Au and semiconductor Cu 2−x Se both played important roles in promoting the production of ROS.

Biocompatibility and Cellular Uptake of Cu 2−x Se/Au-TAT Nanoparticles
The cellular uptake of Cu 2−x Se/Au-TAT nanoparticles in mouse triple-negative breast cancer cell line (4T1) were investigated firstly. As demonstrated in Figure 3A, compared with the free fluorescein isothiocyanate (FITC)-treated group, the obvious fluorescent signals have been observed in both FITC-labeled Cu 2−x Se/Au-TAT-and Cu 2-x Se/Autreated groups, and the fluorescent signal in the FITC-labeled Cu 2−x Se/Au-TAT-treated group was the strongest, indicating that the modification of TAT could enhance the uptake of Cu 2−x Se/Au. Next, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) experiment was employed to test the cytotoxicity of Cu 2−x Se/Au-TAT nanoparticles in 4T1 and human umbilical vein endothelial cell lines (HUVECs). As can be seen from Figure 3B, the cell survival rate of 4T1 and HUVECs cells were still above 80%, though the concentration of Cu 2−x Se/Au-TAT nanoparticles was raised to 120 µg/mL, which demonstrates that the nanoparticles had little cytotoxicity within this concentration range, which lays a good foundation for further experiments.
ticles with or without performing X-ray irradiation.

Biocompatibility and Cellular Uptake of Cu2−xSe/Au-TAT Nanoparticles
The cellular uptake of Cu2−xSe/Au-TAT nanoparticles in mouse triple-negative breast cancer cell line (4T1) were investigated firstly. As demonstrated in Figure 3A, compared with the free fluorescein isothiocyanate (FITC)-treated group, the obvious fluorescent signals have been observed in both FITC-labeled Cu2−xSe/Au-TATand Cu2-xSe/Au-treated groups, and the fluorescent signal in the FITC-labeled Cu2−xSe/Au-TAT-treated group was the strongest, indicating that the modification of TAT could enhance the uptake of Cu2−xSe/Au. Next, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) experiment was employed to test the cytotoxicity of Cu2−xSe/Au-TAT nanoparticles in 4T1 and human umbilical vein endothelial cell lines (HUVECs). As can be seen from Figure 3B, the cell survival rate of 4T1 and HUVECs cells were still above 80%, though the concentration of Cu2−xSe/Au-TAT nanoparticles was raised to 120 μg/mL, which demonstrates that the nanoparticles had little cytotoxicity within this concentration range, which lays a good foundation for further experiments.

Photothermal Ablation and Radiosensitization Effects In Vitro
The photothermal behavior and radiosensitization effects of Cu 2−x Se/Au-TAT nanoparticles were further investigated on 4T1 cells. As can been seen in Figure 3C, the antitumor efficacy of Cu 2−x Se/Au-TAT nanoparticles demonstrated an obvious concentrationdependent way after carrying out an irradiation by 808 nm light. The cell survival rate reduced with the rise of concentrations of Cu 2−x Se/Au-TAT nanoparticles, and the survival rate was only 50% after being cultured with 60 µg/mL Cu 2−x Se/Au-TAT nanoparticles at 1.0 W/cm 2 or 50 µg/mL at 1.5 W/cm 2 . Cell viability even decreased to 80% at 1.0 W/cm 2 or 88% at 1.5 W/cm 2 at 80 µg/mL. The above results demonstrate that Cu 2−x Se/Au-TAT nanoparticles exhibited excellent photothermal conversion properties and could be used to ablate tumor cells in vitro. Figure 2E has already shown that Cu 2−x Se, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT nanoparticles could all produce ROS after X-ray irradiation, indicating that these nanoparticles could act as radiosensitizers for the purpose of radiotherapy. In order to further verify the radio-enhancement effect, a plate colony-formation experiment was conducted; 4T1 cells were co-culture with Cu 2−x Se, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT nanoparticles and were then irradiated at a dose of 0, 2, 4, and 6 Gy, respectively. As Figure 3D shows, with the increase in X-ray dose, the cell survival rate of each group showed a downward trend. However, there was a great diversity in the cell livability among different groups ( Figure 3E). The survival rate S(Cu 2−x Se/Au-TAT) < S(Cu 2−x Se/Au) < S(Cu 2−x Se) < S(control), and the calculated sensitization enhancement ratio (SER) values of Cu 2−x Se, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT were 1.76, 2.13, and 2.55, respectively. The cell livability of Cu 2−x Se/Au nanoparticles was lower than that of Cu 2-x Se nanoparticles, indicating the radio-enhancement effect by combining Cu 2−x Se and Au. Cu 2−x Se/Au-TAT nanoparticles had a higher therapeutic effect than Cu 2−x Se/Au nanoparticles, perhaps owing to the higher cellular uptake caused by TAT.

PTT/RT Combination Therapy In Vitro
The above results have proven that Cu 2−x Se/Au-TAT nanoparticles exerted the potential of not only killing tumor cells as a radiosensitizer, but also ablating tumor cells as a photothermal agent. As monotherapy of RT or PTT is not effective in eradicating tumors [42,43], the combination strategy of RT and PTT is a more desirable tool to enhance the treatment efficacy. Thus, we further studied the combined RT/PTT antitumor effect of Cu 2−x Se/Au-TAT nanoparticles in vitro.
ROS generation was tested by utilizing the probe of DCFH-DA. As demonstrated in Figure 4A, obvious higher green fluorescence intensity was found in the groups of Cu 2−x Se/Au-TAT or Cu 2−x Se/Au treated with X-ray and/or NIR, compared with other groups, indicating the potential of Cu 2−x Se/Au as an effective radiosensitizer to raise RT efficacy. The intensity of green fluorescence in the set treated with Cu 2−x Se/Au-TAT under X-ray and NIR was evidently higher than the group treated with Cu 2−x Se/Au-TAT under only X-ray, which may be attributed to the hyperthermia-induced cellular overactive state. When compared with the group treated with Cu 2−x Se/Au followed by NIR and X-ray, the fluorescence intensity of the group treated with Cu 2−x Se/Au-TAT followed by NIR and X-ray was slightly higher, which may be due to the improvement of cellular uptake by TAT.
Furthermore, DNA damage evaluation was conducted by γ-H 2 AX foci detection through confocal microscopy. As can be seen from Figure 4B, the group of Cu 2−x Se/Au-TAT + PTT + RT showed the highest γ-H 2 AX fluorescent spots, which was obviously higher than the group of Cu 2−x Se/Au-TAT+RT. The results of DNA damage were consistent with those of ROS generation detection, which further confirms that all three variables including RT, PTT, and TAT could promote ROS generation, thus resulting in DNA damage in the nucleus.
A cell colony formation assay, which is considered as the "gold standard" for assessment of cancer cells' response to X-ray irradiation, was also carried out [15]. The results of clonogenic assay and clonogenic survival assay are shown in Figure 4C,D. The cell survival fraction reduced to 50% upon the treatment of Cu 2−x Se/Au-TAT + PTT, 29% of Cu 2−x Se/Au-TAT + RT, and 4% of Cu 2−x Se/Au-TAT + PTT + RT, indicating that combining PTT and RT had the best therapeutic effect. In addition to the radiosensitization and photothermal influence of Cu 2−x Se/Au, the presence of TAT led to the raised cellular uptake of nanoparticles and enhancement of the therapeutic effect.

PTT/RT Combination Efficacy of Cu2−xSe/Au-TAT In Vivo
The photothermal performance of Cu2−xSe/Au-TAT nanoparticles in vivo was investigated by irradiating the tumor areas of 4T1 tumor mice using an 808 nm laser after intratumoral injection of PBS and Cu2−xSe/Au-TAT nanoparticles, and the temperature variation was recorded by an NIR camera. As illustrated in Figure 5A,B, after the tumors underwent a 10-min irradiation at 1.5 W/cm 2 , the temperature of the tumor area with the The PTT/RT combination therapy efficacy of Cu 2−x Se/Au-TAT nanoparticles was ulteriorly visually identified by calcein-AM/PI double staining. After receiving various treatments, 4T1 cells presented different intensities of green (referring to live cells) and red (referring to dead cells) fluorescence. As can been seen from Figure 4E, barely no dead cells were found in the control, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT groups without any treatment, or NIR/X-ray-irradiated control groups, while more dead cells were seen in the Cu 2−x Se/Au and Cu 2−x Se/Au-TAT groups treated with NIR or X-ray. Notably, almost all tumor cells were dead in Cu 2−x Se/Au and Cu 2−x Se/Au-TAT treated with NIR and X-ray, clearly indicating the Cu 2−x Se/Au-TAT-induced combined PTT and RT effects.

PTT/RT Combination Efficacy of Cu 2−x Se/Au-TAT In Vivo
The photothermal performance of Cu 2−x Se/Au-TAT nanoparticles in vivo was investigated by irradiating the tumor areas of 4T1 tumor mice using an 808 nm laser after intratumoral injection of PBS and Cu 2−x Se/Au-TAT nanoparticles, and the temperature variation was recorded by an NIR camera. As illustrated in Figure 5A  Furthermore, the PTT and RT combination therapeutic effects of Cu2−xSe/Au-TAT nanoparticles were explored in vivo. After all treatments, the mice were put to death and tumors were obtained ( Figure 6A). The tumor volume curves are shown in Figure 6B,C. No significant differences could be detected in the groups of control, Cu2−xSe/Au, Cu2−xSe/Au-TAT, and control + PTT, except for a slight decrease in tumor in the control + RT group. Mice treated with Cu2−xSe/Au-TAT + RT and Cu2−xSe/Au-TAT + PTT had an inhibitory effect on tumor volume to 70.3% and 86.3%, respectively, when compared to the control group. Tumor volume from mice receiving Cu2−xSe/Au or Cu2−xSe/Au-TAT mediated PTT alone decreased rapidly in the early stage of treatment, but increased gradually in the later stage, demonstrating that PTT alone could not completely ablate the tumor. Meanwhile, when combining PTT and RT, the inhibitory effect of Cu2−xSe/Au-TAT could reach 99.6%, indicating that combined PTT and RT could effectively achieve tumor ablation. Notably, the inhibitory effects of the Cu2−xSe/Au-TAT-related groups were slightly better than those of the Cu2−xSe/Au-related groups, which may be due to the better tissue permeability. To further estimate the therapeutic effect of Cu2−xSe/Au-TAT-enhanced PTT/RT on tumors, tumor tissue sections were carried out by staining with hematoxylin and eosin (H&E) and antibodies against γ-H2AX ( Figure 6F,G). Compared with the control and the groups of Cu2−xSe/Au and Cu2−xSe/Au-TAT, obvious tumor nucleus fragmentation and nuclear shrinkage were observed in the groups of Cu2−xSe/Au-TAT + PTT and Cu2−xSe/Au-TAT + RT, and this situation was further enhanced when receiving both PTT and RT. In the meantime, the group of Cu2−xSe/Au-TAT + PTT + RT dramatically upregulated the expression levels of γ-H2AX, indicating that serious DNA damage was induced by PTT/RT combined treatment. Furthermore, the PTT and RT combination therapeutic effects of Cu 2−x Se/Au-TAT nanoparticles were explored in vivo. After all treatments, the mice were put to death and tumors were obtained ( Figure 6A). The tumor volume curves are shown in Figure 6B,C. No significant differences could be detected in the groups of control, Cu 2−x Se/Au, Cu 2−x Se/Au-TAT, and control + PTT, except for a slight decrease in tumor in the control + RT group. Mice treated with Cu 2−x Se/Au-TAT + RT and Cu 2−x Se/Au-TAT + PTT had an inhibitory effect on tumor volume to 70.3% and 86.3%, respectively, when compared to the control group. Tumor volume from mice receiving Cu 2−x Se/Au or Cu 2−x Se/Au-TAT mediated PTT alone decreased rapidly in the early stage of treatment, but increased gradually in the later stage, demonstrating that PTT alone could not completely ablate the tumor. Meanwhile, when combining PTT and RT, the inhibitory effect of Cu 2−x Se/Au-TAT could reach 99.6%, indicating that combined PTT and RT could effectively achieve tumor ablation. Notably, the inhibitory effects of the Cu 2−x Se/Au-TAT-related groups were slightly better than those of the Cu 2−x Se/Au-related groups, which may be due to the better tissue permeability. To further estimate the therapeutic effect of Cu 2−x Se/Au-TAT-enhanced PTT/RT on tumors, tumor tissue sections were carried out by staining with hematoxylin and eosin (H&E) and antibodies against γ-H 2 AX (Figure 6F,G). Compared with the control and the groups of Cu 2−x Se/Au and Cu 2−x Se/Au-TAT, obvious tumor nucleus fragmen-tation and nuclear shrinkage were observed in the groups of Cu 2−x Se/Au-TAT + PTT and Cu 2−x Se/Au-TAT + RT, and this situation was further enhanced when receiving both PTT and RT. In the meantime, the group of Cu 2−x Se/Au-TAT + PTT + RT dramatically upregulated the expression levels of γ-H 2 AX, indicating that serious DNA damage was induced by PTT/RT combined treatment. In order to evaluate the potential by-effects of these nanomaterials and the curative treatments, the chief organs of the mice, which consisted of heart, liver, spleen, and kidney, were acquired for H&E staining. No significant tissue injury and adverse effects on the main organs of mice were observed ( Figure S2). Simultaneously, no obvious abnormalities were found in the body weight of the Cu 2−x Se/Au-TAT-treated mice ( Figure 6D,E). These results indicate the high biocompatibility of Cu 2−x Se/Au-TAT nanoparticles within tested days.

Cell Lines and Animals
4T1 and HUVECs were furnished by Biovector NTCC (Beijing, China). All cells were cultured with 10% FBS and 1% penicillin/streptomycin with 5% CO 2 at 37 • C. Female BALB/c mice (4-6 weeks old) were supplied by SPF Biotechnology Co., Ltd. (Beijing, China). The mouse breast cancer xenograft model was developed through 100 µL subcutaneous injection of sterile phosphate buffer saline (PBS) (including 1.0 × 10 6 4T1 cells) on the right back of each mouse. When the tumor volume reached ∼100 mm 3 , the mice could be used for the following in vivo experiments. All animal research was approved by the Animal Ethics Committee of Tianjin Medical University, and all procedures were carried out conforming with the Guide for the Care and Use of Laboratory Animals.
3.3. Synthesis of Cu 2−x Se Nanoparticles, Cu 2−x Se/Au Nanoparticles, and Cu 2−x Se/Au-TAT Nanoparticles Hollow Cu 2−x Se nanoparticles were prepared in reference to former methods, with minor modifications [44]. Briefly, 0.1 mmol CuCl 2 ·2H 2 O was directly dissolved in a mixture of 58 mL 2-propanol and 2 mL water, following the addition of 0.2 g PVP and 0.24 mmol of NaOH solution in 1.2 mL 2-propanol. Next, the solution was reacted for 20 min. After that, 0.2 mL N 2 H 4 •H 2 O (35 wt%) was further added at a very slow speed, and the solution was stirred for another 10 min. Orange Cu 2 O sediments were immediately apparent upon adding N 2 H 4 •H 2 O. The obtained Cu 2 O nanoparticles were gained by centrifugation and subsequent water washing.
In the preparation of hollow Cu 2−x Se nanoparticles, 0.1 mmol of Se power and 0.3 mmol of NaBH 4 were first dissolved in 20 mL water under the protection of N 2 , and the mixed solution was stirred for 30 min to acquire the Se 2solution. Then, the above-mentioned double portion of Cu 2 O nanoparticles was added to the resultant Se 2suspension and stirred for 3 h. The obtained Cu 2-x Se nanoparticles were gained by centrifugation and subsequent water washing.
Next, 0.05 M of HAuCl 4 solution was placed into the Cu 2−x Se nanoparticles dropwise with a mole ratio of 1:0.375 (Cu 2−x Se: HAuCl 4 ) to prepare the Cu 2−x Se/Au nanoparticles. The synthetic products were gained by centrifugation and redispersed in water.
To synthesize Cu 2−x Se/Au-TAT nanoparticles, TAT solution was added to the above synthesized Cu 2−x Se/Au solution dropwise refering to a mass ratio of Cu 2−x Se/Au:TAT of 1:1.5, followed by stirring the mixture for 1 h. Finally, Cu 2−x Se/Au-TAT nanoparticles were gained by centrifugation and subsequent water washing.

Characterization
The morphological features of the obtained samples were observed through a HT7700 TEM (Hitachi, Tokyo, Japan). The elemental mapping was recorded by a JEM-2100F instrument (JEOL, Tokyo, Japan). The absorbance of UV-vis-NIR was obtained by employing a UV-1510 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The fluorescence spectrum was acquired by an F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The hydrodynamic size (Dh) and zeta potentials of the samples were obtained by a ZS90 Zetasizer (Malvern, Malvern, UK) (at Cu 2−x Se concentration of 40 µg/mL. XPS was employed to study the valence states of elementary substances by utilizing an ESCALab 250XiSigma Probe instrument (Thermo Fisher Scientific, Waltham, MA, USA). Cu and Au content of the nanoparticles were determined by a Thermo Electron Corporation X7 ICP-OES (Thermo Fisher Scientific, Waltham, MA, USA). A Rad Source RS-2000 Pro (Rad Source Technologies, Atlanta, GA, USA) was performed for radiation.

Photothermal Experiments
To estimate the photothermal performance of Cu 2−x Se/Au-TAT nanoparticles, the aqueous suspensions of nanoparticles with multiple concentrations (0, 20, 40, 80, 120, 160 µg/mL) underwent irradiation with 808 nm light (1.0 W/cm 2 , 10 min). In addition, Cu 2−x Se/Au-TAT nanoparticles (80 µg/mL) underwent irradiation for 10 min at various light powers (0.5, 1.0, and 1.5 W/cm 2 ). The photothermal stability of Cu 2−x Se/Au-TAT nanoparticles (80 µg/mL) was examined by laser ON/OFF assays for 5 cycles. For each cycle, the Cu 2−x Se/Au-TAT aqueous solution underwent irradiation for 10 min with 808 nm light (1.0 W/cm 2 ), following by light shut off to allow the solution to cool down to ambient temperature. The temperatures of the samples were recorded by an FLIR infrared camera (Nashua, NH, USA).
The photothermal conversion efficiency (η) of Cu 2−x Se/Au-TAT was calculated according to the following equations: where h is the heat transfer coefficient, s is the surface area of the container, Tmax is the maximum temperature of the Cu 2−x Se/Au-TAT solution, Tsur is the maximum temperature of the surroundings, Qdis is the quantity of heat generated when the reagent is water, I is the laser power, A is the absorbance of the Cu 2−x Se/Au-TAT solution at 808 nm, m is the mass of the water solution of nanoparticles, c is the specific heat capacity of the water (4.2 J/g), τ is the time constant of the sample, and T and t are the temperature of the sample and the time of the sample during the cooling process, respectively.

Detection of ROS
ROS detection was carried out by employing DCFH-DA as the probe. Firstly, 20 µL of DMSO solution containing DCFH-DA (0.02 M) and 2 mL of sodium hydroxide (0.01 M) were reacted in the absence of light at ambient temperature for 30 min. Afterwards, 18 mL of phosphate buffer (pH 7.2) was added into the above solution to stop the reaction. Next, DCFH was added into the sample solutions of all the groups. Eight groups were set: Control, Cu 2−x Se, Cu 2−x Se/Au, Cu 2−x Se/Au-TAT, Control + X-ray, Cu 2−x Se + X-ray, Cu 2-x Se/Au + X-ray, Cu 2−x Se/Au-TAT + X-ray. The final concentrations of DCFH were set at 10 µM, the final concentrations of Cu 2−x Se were set at 40 µg/mL, and X-ray was administered at 4 Gy. Afterwards, solutions were shaken on the shaker for 2 h. Finally, the fluorescence of the solutions was obtained using an F-7000 spectrofluorometer with an excited wavelength of 488 nm.

Cellular Uptake Experiment
Firstly, Cu 2−x Se/Au-TAT or Cu 2−x Se/Au was labelled with FITC by adding FITC (100 µg/mL) into Cu 2−x Se/Au-TAT or Cu 2−x Se/Au (1 mg/mL) and reacted for 24 h. The exceeded FITC was removed through washing with water. Next, 4T1 cells were planted into 12-well plates (1 × 10 5 per well) and cultured at 37 • C for 24 h. Subsequently, 4T1 cells were incubated with the medium containing free FITC, FITC-labeled Cu 2−x Se/Au-TAT nanoparticles, and Cu 2−x Se/Au nanoparticles for 4 h. After cleaning three times with PBS, the cells were fastened for 10 min, employing 4% paraformaldehyde. Finally, the cell nucleus was stained by DAPI and imaged, employing confocal fluorescence microscopy.

In Vitro Cytotoxicity Study
The cell cytotoxicity of Cu 2−x Se/Au-TAT nanoparticles towards 4T1 cells and HU-VECs was evaluated using the MTT method. Cells were spread evenly in 96-well plates (5 × 10 3 per well) and cultured for twenty-four hours. Then, cells were cocultured with the medium containing Cu 2−x Se/Au-TAT nanoparticles (10,20,40,60,80, and 120 µg/mL, respectively). After 4-h incubation, the cells were cleaned twice, employing PBS, 100 µL of fresh culture was added, and they were then continuously incubated for 20 h. Subsequently, the above cells were incubated for 4 h absent from dark. The medium was then discarded and they continued to be incubated with 100 µL of 1 mg/mL MTT solution. Afterwards, each well of the medium was discarded, 150 µL of DMSO was further added, and the plates then underwent a 5-min shake. The absorbance of each well was detected at 490 nm, employing a microplate reader.
To evaluate the photothermal ablation effects towards cancer cells, cells were planted into 96-well plates (5 × 10 3 per well) at 37 • C overnight and incubated with multiple concentrations of Cu 2−x Se/Au-TAT nanoparticles (30,40,50,60,70, and 80 µg/mL), respectively. After 4 h, the cells underwent irradiation with 808 nm light at 1.0 or 1.5 W/cm 2 for 10 min, were washed two times, employing PBS, and supplemented with 100 µL of fresh DMEM for another 20 h. The cell livability was performed via the standard MTT test.

In Vitro Clonogenic Assay
4T1 cells were spread evenly into 6-well plates with different densities (250, 500, 1000, and 2000 per well) and cultured for 24 h. Next, the culture containing Cu 2−x Se, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT nanoparticles at Cu 2−x Se concentration of 40 µg/mL was added and incubated for 4 h, and then underwent irradiation with X-ray at 0, 2, 4, and 6 Gy, respectively. After washing with PBS and refreshing medium, all the treated groups were further incubated for 7 d. Finally, the above cells were rinsed two times with PBS, underwent 15-min immobilization with methanol, and were further stained using Giemsa dye for 30 min. Colonies covering at least 50 cells were counted, and the survival fraction (SF) was computed by (surviving colonies)/(cells seeded × plating efficiency) to evaluate the effects of different treatments.
SER was defined with the following formulae: where S represents the cell survival fraction at a certain X-ray dose, D is representative of the dose of the irradiated X-ray, D 0 acts as the average lethal dose of radiation representing the sensitivity of cells to X-ray, and N represents the cell's self-healing ability. D 0 and N were acquired by SPSS. SER was calculated as follows: where D q is representative of the quasi-threshold dose, which represents the ability to repair sublethal injury.

In Vitro Detection of ROS Generation
4T1 cells were inoculated on confocal dishes (8 × 10 4 per well). Twelve groups were set as described previously. Firstly, cells were incubated with control, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT nanoparticles at a Cu 2−x Se concentration of 40 µg/mL for 4 h. Cells were then washed three times with PBS and underwent a 20-min incubation with DCFH-DA (10 µM) and Hoechst 33,258 (10 µM) at 37 • C. After being washed three times with PBS and adding 1 mL of PBS, the experimental groups were treated with or without a 10-min NIR irradiation (808 nm, 1.5 W/cm 2 ), followed by treatment with or without X-ray (4 Gy), respectively. Finally, a fluorescence microscope was employed to observe the ROS created within cells

In Vitro DNA Damage Evaluation
To assess DNA double-strand injury, 4T1 cells in logarithmic growth were spread evenly into 12-well plates (8 × 10 4 per well) and cultured for 24 h. Groups were set the same as in the in vitro ROS detection tests. These cells were incubated with control, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT nanoparticles at a Cu 2−x Se concentration of 40 µg/mL for 4 h, and then treatments were performed with or without a 10-min NIR irradiation (808 nm, 1.5 W/cm 2 ) and/or X-ray (4 Gy). Afterwards, these cells were washed three times with PBS, and then fastened for 10 min by 4% paraformaldehyde. Afterwards, the cells underwent a 10-min permeation by 0.2% Triton X-100 and a 1-h blocking with 3% BSA. After that, the cells were incubated with anti-γ-H 2 AX monoclonal antibody (1:250) at 4 • C overnight, followed by a treatment of secondary Alexa-488-conjugated goat anti-rabbit antibody (1:300). Finally, the cell nuclei underwent a 10-min staining by DAPI (1:900) and were imaged, employing confocal fluorescence microscopy.

Live-Dead Cell-Staining Assay
4T1 cells were inoculated in 12-well plates (1 × 10 5 per well) and incubated for 24 h. Then, these cells were incubated with control, Cu 2−x Se/Au, and Cu 2−x Se/Au-TAT nanoparticles at a Cu 2−x Se concentration of 60 µg/mL for 4 h. Following the various treatments mentioned above, a calcein-AM/PI double staining kit was employed to stain for 30 min to make a distinction between the live cells (green) and the dead cells (red). Finally, a Zeiss fluorescence microscope (Jena, Germany) was employed to observe the acquired results.

In Vivo Antitumor Efficacy
The 4T1 tumor-bearing mice were distributed into twelve groups of 6 at random, as described above, as long as the tumor grew to 100 mm 3 . The twelve groups of mice were then intratumorally injected with 50 µL control, Cu 2−x Se/Au (at a Cu 2−x Se concentration of 4 mg/mL), and Cu 2−x Se/Au-TAT nanoparticles (at a Cu 2−x Se concentration of 4 mg/mL), respectively. The mice of different groups then underwent irradiation with or without a 10-min NIR light (808 nm, 1.5 W/cm 2 ) and/or X-ray (6Gy). The volume of tumors and the mouse weight was noted every second day during the whole treatment period. The volume of tumors was measured referring to the following computing formula: volume = 1/2(width 2 × length). All tumors were imaged on the 20th day after treatments.

Histology Analysis In Vivo
The chief organs, which consisted of heart, liver, spleen, and kidney, were acquired for histology analysis after various treatments. These removed organs were fastened with 4% paraformaldehyde and were further made into paraffin sections for H&E staining. Additionally, the tumors were obtained after treatments for 48 h, fastened by 4% paraformaldehyde, and further embedded in paraffin. Slices were stained by H&E and γ-H2AX antibody. All these images of tissue slices were imaged, employing a fluorescence microscope.

Statistical Analysis
All the above results were assessed via Student's t-test or one-way ANOVA. The experimental results were given as mean ± standard deviation (SD). p < 0.05 was representative of statistical significance.

Conclusions
In conclusion, multifunctional Cu 2−x Se/Au-TAT nanoparticles were prepared by a spontaneous redox process between hollow Cu 2-x Se nanoparticles and HAuCl 4 , with subsequent surface modification with TAT. They exhibited excellent stability, enhanced tumor cell uptake, excellent radiosensitization, and outstanding photothermal properties. Both in vitro and in vivo, compared with PTT or RT monotherapy, combination therapy of PTT and RT achieved a significant combined anticancer effect. This study provided an efficient mentality for RT/PTT combination therapy against breast cancer.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/molecules28010423/s1, Figure S1: XPS survey spectra of (A) Cu 2−x Se/Au nanoparticles and (B) Cu 2-x Senanoparticles; (C) Cu and (D) Se spectra of Cu 2−x Se nanoparticles. Figure  S2: H&E staining images of tissue sections from the mice after different treatments.
Author Contributions: Conceptualization, R.R., Y.W. and X.Y.; methodology, S.G. and R.R.; validation, R.R., S.G., X.J. and Z.Q.; investigation, R.R. and S.G.; resources, S.G. and Z.G.; writing-original draft preparation, R.R.; writing-review and editing, X.Y., Y.W. and M.C.; supervision, M.C.; project administration, X.Y.; funding acquisition, R.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: The animal study protocol was approved by the university animal care and use committee of Tianjin Medical University.

Informed Consent Statement: Not applicable.
Data Availability Statement: Written informed consent has been obtained from the patient(s) to publish this paper.