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Scintillator-based Nanohybrids with Sacrificial Electron Prodrug for Enhanced X-ray-induced Photodynamic Therapy Han Wang, Bin Lv, Zhongmin Tang, Meng Zhang, Weiqiang Ge, Yanyan Liu, Xinhong He, Kuaile Zhao, Xiangpeng Zheng, Mingyuan He, and Wenbo Bu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02409 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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Scintillator-based Nanohybrids with Sacrificial Electron Prodrug for Enhanced X-ray-induced Photodynamic Therapy Han Wang[1,3], Bin Lv[4], Zhongmin Tang[1,3], Meng Zhang[1,3], Weiqiang Ge[4], Yanyan Liu[2], Xinhong He[5], Kuaile Zhao[5], Xiangpeng Zheng[4], Mingyuan He[2], and Wenbo Bu*[1,2] [1] State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China [2] Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China [3] University of Chinese Academy of Sciences, Beijing 100049, China [4] Department of Radiation Oncology, Shanghai Huadong Hospital, Fudan University, Shanghai 200040, China [5] Department of Radiology, Shanghai Cancer Hospital, Fudan University, Shanghai 200032, China

ABSTRACT X-ray-induced photodynamic therapy (X-PDT) has high depth of penetration and has considerable potential for applications in cancer therapy. Scintillators and heavy

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metals have been adopted to absorb X-rays and transmit the energy to photosensitizers. However, the low efficiency of converting X-rays to reactive oxygen species (ROS) presents a challenge for the use of X-PDT to cure cancer. In this study, a new method based on LiLuF4:Ce@SiO2@Ag3PO4@Pt(IV) nanoparticles (LAPNP) is presented that could be used to enhance the curative effects of X-PDT. To make full use of the fluorescence produced by nanoscintillators (LiLuF4:Ce), a cisplatin prodrug Pt(IV) was utilized as a sacrificial electron acceptor to increase the yield of hydroxyl radicals (·OH) by increasing the separation of electrons and holes in photosensitizers (Ag3PO4). Additionally, cisplatin is produced upon the acceptance of electrons by Pt(IV) and further enhances the damage caused by ·OH. Via a two-step amplification, the potential of LAPNP to enhance the effects of X-PDT has been demonstrated.

KEYWORDS: Cancer, photodynamic therapy, photosensitizer, sacrificial electron acceptor, reactive oxygen species

Cancer is an enormous threat to the human population, and more effective therapeutic methods are urgently needed.[1] Photodynamic therapy (PDT) has attracted considerable attention because of its temporal and spatial selectivity. Photosensitizers and light sources are necessary in PDT. Upon excitation by ultraviolet light (UV) and visible light (Vis), photosensitizers facilitate the transformation of oxygen into singlet oxygen molecules that, are destructive to biomacromolecules.[2] However, there are two notable disadvantages of this technique. First, the penetration depth of UV and Vis into biological tissue is only several micrometers, which limits the clinical application of PDT.[3] Second, there is little oxygen inside hypoxic tumors, which will lead to the production of inadequate amounts of singlet


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oxygen and thereby limit the usefulness of the therapy.[4] To increase the efficacy of PDT in the treatment of cancer, further research is required to develop methods to overcome these disadvantages. With the advent of nanomedicine, considerable progress has been made in counteracting the limits of PDT. First, X-ray-induced photodynamic therapy (X-PDT) has received considerable attention recently because X-rays have a high penetration depth in the human body. To overcome the energy mismatch between X-rays (keV-MeV) and the light absorbed by photosensitizers (eV), scintillators and heavy metals have been adopted to absorb X-rays and transmit the energy to photosensitizers.[5,

6]

Second, in order to circumvent the

dependence of PDT on the presence of oxygen, our group has made progress in using nanosemiconductors as photosensitizers.[7,

8]

Upon the irradiation of nanosemiconductors

with light, electrons and holes appear in pairs. The holes react with water to produce hydroxyl radicals (·OH), which can kill cancer cells with high efficiency.[9] Therefore, the use of X-PDT with nanosemiconductors will likely be able to overcome the disadvantages of PDT in its current form. However, due to the low conversion efficiency of nanoscintillators, the effects of X-PDT is limited.[7,10] Considering the enormous challenges presented by increasing the energy deposited by X-rays, an improvement in the efficiency of photosensitizers is a more feasible and appropriate way to increase the effects of X-PDT. Surprisingly, little research has been conducted towards this goal. In this study, we developed a new method to increase the effects of X-PDT via the introduction of sacrificial electron acceptors that have been widely used in other fields, such as photocatalysis.[11] When the semiconductors are excited, electrons and holes will not only appear in pairs but also recombine; thus, little ·OH will be produced. However, upon the addition of sacrificial electron acceptors, the electrons will be accepted, and the holes will separate from the electrons, leading to an increase in the yield of ·OH. In this study, a


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cisplatin prodrug (Pt(IV)) was utilized as a sacrificial electron acceptor due to its oxidizability in order to separate the electrons and holes. Coincidently, cisplatin is produced when Pt(IV) accepts electrons, and cisplatin can attack the deoxyribonucleic acid (DNA) in cancer cells and thereby enhance the effect of X-PDT.[12] Although only a small amount of the X-ray energy can be deposited in the nanoscintillators, the addition of Pt(IV) can increase both the yield and the effects of ·OH, which will fully unlock the potential of nanosemiconductors and increase the efficacy of X-PDT.

Scheme 1. Diagram showing the preparation process and mechanisms underlying the effects of X-PDT with LiLuF4:Ce@SiO2@Ag3PO4@Pt(IV) nanoparticles (LAPNP).

The process of preparing the materials and the mechanisms underlying their action are illustrated in Scheme 1. First, LiLuF4:Ce nanoparticles were chosen for use as the scintillator and obtained by pyrolysis.[13] LiLuF4:Ce with a tetragonal phase, as confirmed by transmission electron microscopy (TEM) (Figure 1a), was successfully obtained. For the


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combination of LiLuF4:Ce and Ag3PO4, LiLuF4:Ce was coated with SiO2 and later modified with amino groups. The thickness of the SiO2 coating of LiLuF4:Ce@SiO2 was 10 nm (Figure 1b). Second, Ag3PO4 nanoparticles were used as the nanosemiconductor; this finding represents the first report of their use for PDT.[14] Ag3PO4 nanoparticles were prepared using the reverse microemulsion method, which is an older technique.[15] Upon mix of the reverse microemulsions of AgNO3 and Na2HPO4 (Figure 1c), Ag3PO4 nanoparticles with a size of 3 nm were produced (Figure 1d). Interestingly, the Ag3PO4 nanoparticles preferred to form a row. Changing the concentrations of the compounds used in the reverse microemulsions allowed for the easy regulation of the size of the Ag3PO4 nanoparticles, with nanoparticles of different sizes being obtained (Figure S1a and S1b, Supporting Information). Third, Pt(IV) was used to separate the light-induced electrons and holes. Fourier transform infrared (FT-IR) (Figure S2a, Supporting Information) and 1H NMR (Figure S2b, Supporting Information) spectroscopy were used to characterize the Pt(IV), and the results conformed to those obtained from previous research.[16] A KI solution was applied to the Pt(IV) and reacted to generate rufous iodine as shown in Figure S2c (Supporting Information). This experiment verified the oxidizability of Pt(IV), which was the source of its capacity to accept electrons (as detailed in the Supporting Information). As silver is strongly coordinated with amino and carboxyl groups, Ag3PO4 can attach to the surface of LiLuF4:Ce@SiO2 to produce LiLuF4:Ce@SiO2@Ag3PO4 (denoted LANP), and Pt(IV) (contain carboxyl groups) can then attach to the LANP surface to produce LiLuF4:Ce@SiO2@Ag3PO4@Pt(IV) (denoted LAPNP).[17] First, LANP and LAPNP were characterized using TEM (Figure S1c, Supporting Information and Figure 1e), which showed that small Ag3PO4 nanoparticles were interspersed on the surface of the LiLuF4:Ce@SiO2. Xray diffraction (XRD) patterns produced by LAPNP (Figure 1f) indicated the presence of LiLuF4:Ce and Ag3PO4. To further characterize LAPNP, the FT-IR and UV-Vis absorption of


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LAPNP (Figure S3a and Figure S3b, Supporting Information) were measured. As shown in Figure S3a, the characteristic peaks of Si-O-Si and PO43- were produced by LAPNP. [18] The hydrodynamic size and zeta potentials of LANP and LAPNP were also measured. As presented in Figure S4a (Supporting Information), the hydrodynamic size of LANP was considerably larger than that of LAPNP. In other words, LANP easily coagulated in solution, but LAPNP was more stable. The zeta potential of LAPNP was -49 mV due to ionization of the carboxyl group of Pt(IV) (Figure S4c, Supporting Information). However, without the protection of Pt(IV), LANP had a zeta potential of only 3 mV. The enormous difference in the zeta potential led to a difference in their hydrodynamic sizes. Finally, to ascertain the elemental composition of LAPNP, X-ray photoelectron spectroscopy (XPS), element mapping images of scanning electron microscope (SEM) and inductively coupled plasma optical emission spectrometry (ICP-OES) were used. XPS showed that Pt was present only on the surface of LAPNP (Figure S4b, Supporting Information), while Ag was present on the surfaces of both LANP and LAPNP (Figure S4d, Supporting Information), which showed that Pt(IV) was successfully incorporated onto the surface of LAPNP. Element mapping images of SEM (Figure S5, Supporting Information) demonstrated the presence of P, Pt and Ag on LAPNP, with the total amount of Pt being considerably less than that of Ag. The mass concentrations of Lu, Ce, Ag, and Pt on LAPNP were approximately 60%, 2.5%, 5%, and 1%, respectively, which correlated well with the data obtained from the element mapping images. These experiments and the resulting data confirmed the successful preparation of LAPNP and its superior dispersibility and stability to that of LANP due to the incorporation of Pt(IV).


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Figure 1. Characterization of materials. a) TEM graph of LiLuF4:Ce. b) TEM graph of LiLuF4:Ce@SiO2. c) Photograph of reverse microemulsion with the left panel showing AgNO3 and the right showing Na2HPO4. d) TEM graph of Ag3PO4 nanoparticles. e) TEM graph of LAPNP. f) XRD patterns of LAPNP.

After the successful preparation of LAPNP, the performance of X-PDT was measured in solution. First, the fluorescence spectrum of LiLuF4:Ce and the absorption spectrum of Ag3PO4 (Figure 2a) were characterized. Upon X-ray irradiation (80 kV), the fluorescence peaks of LiLuF4:Ce occurred at 305 nm and 325 nm due to the 5d to 4f transition of the cerium atoms.[13] For Ag3PO4, the absorption peak usually occurs at 350 nm.[14] However, during this study, the absorption peak of Ag3PO4 occurred at 295 nm due to the widening of the energy bandwidth caused by the nanomaterials. Nevertheless, the absorption of Ag3PO4


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was still compatible with the fluorescence of LiLuF4:Ce. Second, the ability of LAPNP to produce ·OH upon irradiation with X-rays (6 MeV) in solution was measured. The pattern of the electron spin resonance (ESR) spectrum of LAPNP in the presence of X-rays (Figure 2b, Figure S6 in Supporting Information) demonstrated the generation of ·OH. [19] The amount of ·OH that was produced was estimated by the measurement of the degradation of rhodamine B (RhB).[20] Figure 2c indicated that LAPNP had the strongest ability to produce ·OH in solution, with more than 30% of the RhB being degraded in the presence of 20 Gy X-rays. Compared to LAPNP, LANP had a lower production capacity, with 20% RhB degradation. In contrast, the efficiency of Pt(IV) was not greatly different from that of the control group, and approximately 10% of the RhB was degraded in both groups. X-rays are a type of ionizing radiation and will therefore interact with water to produce ROS which play an important role in radiation therapy. Hence, even without the presence of any materials, a portion of RhB was still degraded. Upon the addition of LANP and LAPNP, more of the Xray energy was deposited due to the presence of heavy elements. However, more energy deposition does not necessarily correspond to a higher ·OH yield. For LANP, which has a low efficiency in its separation of electrons and holes, energy is more likely to be dissipated to the environment via electron-hole recombination. In contrast, the use of LAPNP, which contains Pt(IV), will lead to the separation of more holes and, consequently, the production of more ·OH. These observations may explain why LAPNP causes the most degradation of RhB, while LANP has worse performance. Finally, the release of cisplatin from LAPNP after Xray exposure (6 MeV) was characterized. As shown in Figure S7 (Supporting Information), the new signals generated by LAPNP in the presence of X-rays compared with those generated by LAPNP during

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Pt NMR demonstrated the generation of cisplatin.[21] In

Figure 2d, the calculated amount of released cisplatin is shown while Figure 2c and Figure 2d


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show that the greater the dosage of X-ray irradiation is, the greater the amount of ·OH and cisplatin by LAPNP is.[22]

Figure 2. Characterization of materials. a) X-ray (80 kV) induced fluorescence spectrum of LiLuF4:Ce and UV-Vis absorption spectrum of Ag3PO4. b) Electron spin resonance (ESR) spectrum of LAPNP (1000 μg/ml) in the presence of 0 Gy and 4 Gy of X-rays (6 MeV); the dose rate was 4 Gy/min. c) Degradation of RhB (5 μg/ml) in the presence of saline (control), Pt(IV) (20 μg/ml), LANP (1000 μg/ml), LAPNP (1000 μg/ml) and 0 Gy, 5 Gy, 10 Gy, 15 Gy and 20 Gy of X-rays (n=5, 6 MeV); the dose rate was 4 Gy/min. The amount of RhB was measured by absorbance at λ=554 nm. d) Release of platinum from LAPNP (1000 μg/ml) in the presence of 0 Gy, 5 Gy, 10 Gy, 15 Gy and 20 Gy of X-rays (n=5, 6 MeV); the dose rate was 4 Gy/min.

To further demonstrate the effects of X-PDT on LAPNP, in vitro experiments were conducted on HeLa cells in the presence of 6 MeV X-rays. First, the cytotoxicity of LAPNP in HeLa and LO2 (normal human liver) cells was determined. As presented in Figure S8a and S9a (Supporting Information), there was no obvious cytotoxicity at a concentration of 20


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μg/ml, which was used for all of the in vitro experiments. Only a small amount of free Ag and Pt was found to leach into solution (Figure S8b, Supporting Information), demonstrating the stability of LAPNP. The cytotoxicity of Pt(IV) and cisplatin was also measured. Pt(IV) was less cytotoxicity than cisplatin, as shown in Figure S9b (Supporting Information), which conformed to the results found in other studies.[16, 23] LAPNP can be taken up by HeLa cells and enters via lysosomes, as observed by confocal microscopy (Figure S9c, S10a and S10b, Supporting Information). Second, the yield of ·OH was determined using hydroxyphenyl fluorescein (HPF), an intracellular probe that can detect ·OH. As shown in Figure 3, little ·OH was detected in any of the groups in the absence of X-ray irradiation. In contrast, in the presence of X-rays, both LANP and LAPNP produced ·OH in normoxic cells, with LAPNP producing a higher yield, in line with earlier experiments conducted in solution. However, only LAPNP produced ·OH efficiently in hypoxic cells, with LANP producing little ·OH. Two factors led to this result, the first being the well-known observation that Xrays are not very lethal to hypoxic cells due to the abnormally high ability of these cells to repair DNA and scavenge free radicals.[24] Thus, a fraction of the ·OH will be scavenged by the hypoxic cells themselves and will not be detected by HPF. The second factor is the fact that, although several reports have claimed that semiconductor photosensitizers can function without oxygen, the presence of oxygen will increase the yield of ·OH by accepting electrons and separating holes.[7] Pt(IV) functions in LAPNP as a sacrificial electron acceptor, so oxygen can only slightly improve the ·OH production of LAPNP. Conversely, there are no sacrificial electron acceptors in LANP, then the presence of oxygen becomes more important for the ·OH yield.


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Figure 3. Detection of ·OH in vitro. The dose rate was 4 Gy/min and the energy of the Xrays was 6 MeV. Upon irradiation with 0 Gy and 4 Gy of X-rays, the yield of ·OH in normoxic cells or hypoxic cells co-cultured with LANP (20 μg/ml), LAPNP (20 μg/ml) or saline (control group) was characterized. The fluorescence intensity of HPF correlated with the yield of ·OH. The scale bar represents 50 μm.

Many studies have demonstrated that ·OH damages cells by causing double-strand breaks in DNA.[25] Moreover, in this study, Pt(IV) can be converted to cisplatin, which can enhance the level of DNA damage caused by ·OH.[26] To verify this enhancement, DNA damage was measured by staining of γ-H2AX, as shown in Figure 4a and S11a (Supporting Information).[27] Without X-rays, no DNA damage occurred. Upon X-ray irradiation, there was less DNA damage in the presence of LANP than LAPNP, despite the fact that LANP produced ·OH in normoxia without the aid of Pt(IV). In addition, LANP caused little DNA damage in hypoxia, while damage still occurred in the presence of LAPNP. DNA damage was also measured by single cell electrophoresis, and the results (Figure S12, Supporting Information) validated the results of the γ-H2AX staining. These data indicated that both ·OH and Pt(IV) played important roles in the effects of X-PDT.


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Due to the performance of LAPNP in normoxia and hypoxia, cell apoptosis and proliferative injury were also characterized. First, a cell colony formation assay was carried out to demonstrate proliferative injury in cells. As presented in Figure 4b, 4c, S11b and S11c (Supporting Information), both normoxic cells and hypoxic cells exposed to LAPNP and Xrays had the least proliferation compared to the other groups, especially in the group exposed to 6 Gy of X-rays in the presence of LAPNP, in which almost no cells survived. Using a multi-target and single-hit model, the sensitive enhancement ratios (SER) of LANP and LAPNP were calculated (details in the Supporting Information).[28] For normoxic cells, the SER of LANP was 1.05, while the SER of LAPNP was 1.24. In contrast, for hypoxic cells, the SER of LANP was 1.00, while the SER of LAPNP was 1.28. Second, a flow cytometer was used to measure the apoptosis of HeLa cells. In line with the results of the cell colony formation assay, LAPNP caused the most cell apoptosis in the presence of X-ray irradiation in both normoxic and hypoxic cells (Figure S13, Supporting Information). Third, cell viability assays were also carried out during this study. Figure S14 (Supporting Information) shows the effects of PDT with Ag3PO4 in the presence of UV irradiation (350 nm, 0.1 W/cm2, 2 min). Figure S15 (Supporting Information) shows that LAPNP in the presence of X-rays caused the most cell death in both normoxic and hypoxic cells, which validated the results shown in Figure 4b, 4c and S13. As demonstrated by the results of all the experiments performed in vitro, the addition of Pt(IV) allowed LAPNP to produce sufficient ·OH in the presence of X-ray irradiation, and the cisplatin generated from Pt(IV) enhanced the level of DNA damage caused by ·OH. Additionally, substantial cell apoptosis and proliferative injury was shown to occur.


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Figure 4. X-PDT in vitro. The dose rate was 4 Gy/min and the energy of the X-rays was 6 MeV. a) Detection of DNA damage in vitro. Upon irradiation with 0 Gy and 4 Gy of X-rays, the DNA damage in normoxic or hypoxic cells co-cultured with LANP (20 μg/ml), LAPNP (20 μg/ml) or saline (control group) was characterized. The level of fluorescence of γ-H2AX correlated with the DNA damage. Scale bar represents 50 μm. b) and c) Detection of the rate of cell colony formation. Upon irradiation with 0 Gy, 2 Gy, 4 Gy and 6 Gy of X-rays (n=3), the rate of cell colony formation in normoxic (b) or hypoxic cells (c) co-cultured with Pt(IV) (0.4 μg/ml), LANP (20 μg/ml), LAPNP (20 μg/ml) or saline (control group) was calculated.

Because LAPNP provided a curative effect in vitro, the performance of LAPNP was evaluated in vivo. The high biocompatibility of LAPNP was proved by comparing the blood parameters (Figure S16, Supporting Information), hematoxylin and eosin (H&E) staining of major organs (Figure S17, Supporting Information) and weights (Figure S18a and S18b,


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Supporting Information) of the LAPNP group to those of the control group. Next, tumorbearing mice were used to demonstrate the effects of X-PDT. To avoid the influence of individual differences, two tumors were implanted in each mouse. The tumor on the right was irradiated with X-rays, while the tumor on the left was not irradiated (Figure 5b). The use of tangential radiation field (1.5 cm×1.5 cm, Figure 5b) can ensure that the whole tumor is exposed to X-rays with little exposure of normal tissue. Prior to X-ray irradiation, LANP, LAPNP and saline were injected intratumorally. The change in the tumor volumes of each group is presented in Figure 5a and S19 (Supporting Information). After irradiation, the growth of the tumors treated with LAPNP was suppressed, while the tumors in the other groups developed rapidly. As shown by H&E and TdT-mediated dUTP nick end labeling (TUNEL) staining, a larger apoptotic area was found in the tumors treated with LAPNP and X-ray, indicating that the structures of the cancer cells was damaged (Figure 4c). In contrast, the damage was found to be slight in the presence of LANP. These data clearly indicate that LAPNP has a considerably better curative effect than LANP, validating the experiments performed in vitro.


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Figure 5. X-PDT in vivo. The dose rate was 4 Gy/min and the energy of the X-rays was 6 MeV. a) Volume of tumors treated with 0 Gy and 4 Gy of X-rays after intratumoral injection of saline (100 μl), LANP (1000 μg/ml,100 μl), or LAPNP (1000 μg/ml,100 μl) (n=5, ***P

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