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Perfluorocarbon-Loaded Hollow Bi2Se3 Nanoparticles for Timely Supply of Oxygen under Near-Infrared Light to Enhance the Radiotherapy of Cancer Guosheng Song, Chao Liang, Xuan Yi, Qi Zhao, Liang Cheng, Kai Yang, and Zhuang Liu*
Radiation therapy (RT) is a widely used cancer treatment strategy, in which the ionizing radiation (e.g., X-ray, γ-ray) locally applied on the tumor is able to generate oxygen-centered radicals formed by the ionization of surrounding water to induce DNA damage and thereby kill cancer cells.[1] Especially, if oxygen is available, it can react with the broken ends of DNA to create stable organic peroxides, which cannot be easily repaired, greatly enhancing the degree of RT-induced cellular damage.[1a,c,d] However, during RT, normal cells under exposure to the radiation beam would also be nonselectively killed, resulting in severe toxic side effects.[2] On the other hand, since the degree of cellular damage caused by ionizing radiation is highly dependent on the level of cellular oxygenation,[1a] while the tumor microenvironment is more hypoxic than normal tissues, hypoxia-associated resistance of RT often occurs, particularly for cancer cells in the center of a tumor where the oxygen supply is deficient.[3] In recent years, various strategies including nanomedicine approaches have been explored to improve the therapeutic outcomes of RT in cancer treatment.[4] Many efforts have been devoted to the development of chemoradiotherapy by using nanoparticle-drugs to enhance the efficacy of RT.[5] It has also been found that tumor-homing nanoparticles containing heavy elements which exhibit strong X-ray attenuation ability may be used to sensitize RT by concentrating a great deal of irradiation energy inside the tumor, so as to enhance the efficacy and specificity of RT in cancer treatment.[5b,6] Moreover, several methods have been developed to overcome the hypoxia-associated RT resistance by challenging the hypoxic tumor microenvironment, such as the use of MnO2 nanoparticles, bioreductive pro-drugs (e.g., tirapazamine), as well as X-ray-controlled NOreleasing upconversion nanoparticles.[5c,7] In our recent work, it was found that the mild photothermal effect induced by nearinfrared (NIR)-light-absorbing nanoparticles could be utilized Dr. G. Song, C. Liang, Prof. L. Cheng, Prof. Z. Liu Institute of Functional Nano & Soft Materials (FUNSOM) Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices Soochow University Suzhou, Jiangsu 215123, China E-mail: [email protected] X. Yi, Q. Zhao, Prof. K. Yang School of Radiation Medicine and Protection & School for Radiological and Interdisciplinary Sciences (RAD-X) Medical College of Soochow University Suzhou, Jiangsu 215123, China
DOI: 10.1002/adma.201504617
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to increase intratumoral blood flow and subsequently improve the oxygenation status of tumor microenvironment, so as to enhance the therapeutic efficacy of RT.[5a] Perfluorocarbon (PFC) is widely used in ultrasound contrast imaging and its several gaseous formulations are commercially available for diagnostic imaging.[8] Moreover, liquid PFC can dissolve a large amount of oxygen and serve as an oxygen carrier, enabling it to be used as a blood substitute.[8a,b,9] The PFC emulsions (such as Fluosol-DA- 20%) have been approved for intravenous use by the United States Food and Drug Administration.[10] In this work, hollow Bi2Se3 nanoparticles, which are able to absorb both NIR light and X-ray irradiation, are prepared by a facile cation exchange method and then functionalized with polyethylene glycol (PEG). Interestingly, those nanoparticles with the hollow structure could be effectively filled with perfluorohexane (a kind of liquid PFC), which then is able to act as an oxygen reservoir. After being saturated with oxygen, the obtained PEG-Bi2Se3@PFC@O2 can gradually release oxygen and offer significantly increased radiation-induced DNA damage of cancer cells during RT, in comparison to that achieved with PEG-Bi2Se3 alone. Importantly, we further uncover that exposure of PEG-Bi2Se3@PFC@O2 to NIR light would result in the burst release of oxygen from nanoparticles and thus the instantly increased tumor oxygenation, which is greatly helpful to overcome the hypoxia-associated RT-resistance. Since Bi2Se3 by itself could also sensitize RT by concentrating irradiation energy in the tumor, RT treatment of cancer with PEG-Bi2Se3@ PFC@O2 under enhancement with NIR light is found to be extremely effective in preventing the tumor growth. In our previous report, a cation exchange method was used to produce MnSe@Bi2Se3 core-shell nanostructures from MnSe nanoparticles with a relatively large size (average length of ≈132 nm and width of ≈105 nm) at the temperature of 180 °C.[5a] It is hypothesized that if a smaller template with a high surface-to-volume ratio is used, the rate of ions outward diffusion should be dramatically increased than that of inward diffusion, resulting in a cavity within the nanoparticle due to the nanoscale Kirkendall effect.[11] Herein, smaller MnSe nanoparticles were used as templates, starting from which hollow Bi2Se3 nanoparticles were prepared by a facile one-pot cation exchange method and Kirkendall effect (Figure 1a). In brief, MnSe nanoparticles with the average diameter of ≈35 nm as templates were pre-made (Figure S1, Supporting Information)[12] and then added with bismuth source under heating. The higher pKsp value of bismuth chalcogenides than manganese chalcogenides would be favorable to promote cation exchange of manganese by bismuth.[13]
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COMMUNICATION Figure 1. Synthesis and characterization of hollow Bi2Se3 nanoparticles. a) A scheme showing the preparation of hollow Bi2Se3 nanoparticles using MnSe nanoparticles as the template. b) TEM images of core-shell MnSe@Bi2Se3 prepared at 140 °C (Inset: the corresponding magnified TEM image). c) HAADF-STEM image of core-shell MnSe@Bi2Se3 prepared at 140 °C. The element maps showed the distribution of Mn, Se, and Bi. d) TEM images of hollow Bi2Se3 nanoparticles prepared at 180 °C (Inset: the corresponding magnified TEM image). e) EDX spectrum of hollow Bi2Se3 prepared at 180 °C. No Mn signal was found in the EDX spectrum of those hollow nanostructures. f) UV–vis–NIR absorbance spectra of PEG-Bi2Se3 nanoparticles in aqueous dispersions with various concentrations. g) Temperature elevation curves of aqueous solutions containing PEG-Bi2Se3 with different concentrations, under the irradiation of an 808 nm laser (0.8 W cm−2).
We carefully evaluated how the heating temperature would affect the final nanoparticle structure. It has been found that when the reaction temperature reached 120 °C (Figure S2a, Supporting Information) and 140 °C (Figure 1b and Figure S2b, Supporting Information), Mn2+ would be partially exchanged by Bi3+ in the outer layer of nanoparticles, resulting in coreshell MnSe@Bi2Se3 nanoparticles as evidenced by transmission electron microscope (TEM) images (Figure 1b inset and Figure S2b inset, Supporting Information), the high-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping (Figure 1c), as well as X-ray diffraction (XRD) data (Figure S3, Supporting Information). With further increase of temperature from 160 °C (Figure S2c, Supporting Information) to 180 °C (Figure 1d and Figure S2d, Supporting Information), the core–shell nanostructure gradually transformed into the hollow Bi2Se3 structure. The resulted hollow Bi2Se3 nanoparticles showed a well-maintained morphology
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of MnSe templates with the uniform size of ≈35 nm and shell thickness of ≈8 nm. As expected, magnification of TEM images (Figure 1d inset and Figure S2b inset, Supporting Information), energy dispersive spectra (EDX) (Figure 1e), and XRD data (Figure S3, Supporting Information) further confirmed the successful fabrication of hollow Bi2Se3. Therefore, using small MnSe nanoparticles as the template, increase of temperature could firstly activate the cation exchange to form core–shell MnSe@Bi2Se3, and then promote the transformation of such core–shell nanoparticles into the hollow Bi2Se3 nanostructure, probably due to the outward diffusion of manganese ion accelerated by the nanoscale Kirkendall effect.[11,14] After transformation into hollow Bi2Se3, the specific surface area of those nanoparticles was measured to be 76.3 cm2 g−1 (Figure S4, Supporting Information), allowing the effectively loading of guest molecules in the inner cavity of such hollow nanostructure.
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Figure 2. Oxygen-loaded nanoparticles for enhanced radiotherapy. a) A scheme showing hollow PEG-Bi2Se3 nanoparticles with PFC loading as an oxygen carrier. b) O2 concentration changes after addition of PEG-Bi2Se3@O2 or PEG-Bi2Se3@PFC@O2 into deoxygenated water under a nitrogen atmosphere. c) γ-H2AX-stained 4T1 cells treated with PBS, PEG-Bi2Se3, RT, PEG-Bi2Se3 + RT, or PEG-Bi2Se3@PFC@O2 +RT, under a hypoxic condition. The nanoparticle concentration was 100 µg mL−1 and the irradiation dose was 6 Gy. Cells exposed to PEG-Bi2Se3@PFC@O2 nanoparticles after RT exhibited the highest level of DNA damage (positive in γ-H2AX). d) Quantitative analysis of γ-h2ax foci density (γ-h2ax foci/100 µm2) for n > 100 cells in each treatment group. P values: **P < 0.01, by analysis of variance (ANOVA).
A layer-by-layer (LBL) polymer coating method was then developed to modify those hollow Bi2Se3 nanoparticles,[15] whose surface was covered by oleylamine post-synthesis. Bi2Se3 nanoparticles were firstly modified with dimercaptosuccinic acid (DMSA), which would offer those nanoparticles negative charges and make them dispersible in water. Such DMSAmodified nanoparticles were then encapsulated with cationic poly(allylamine hydrochloride) (PAH) and anionic poly(acrylic acid) (PAA) in sequence. After further conjugation with amineterminated PEG (NH2-PEG, Mw = 5000), the resulted PEGylated hollow Bi2Se3 (PEG-Bi2Se3) showed great dispersity in various physiological solutions. The successful LBL coating was confirmed by the alternative change of zeta potentials (Figure S5a, Supporting Information) and gradual increase of hydrodynamic sizes (Figure S5b, Supporting Information) of those nanoparticles after different layers of polymers were coated. Moreover, PEG-Bi2Se3 possessed strong absorbance in NIR region (Figure 1f), and thus exhibited an outstanding photothermal effect upon irradiation by an 808 nm NIR laser (Figure 1g). PFC can dissolve a large amount of oxygen due to the van der Waals interaction between PFC and oxygen.[16] Previous studies have demonstrated that PFC could be infused into the cavity of various hollow nanostructures (hollow SiO2, hollow Prussian 2718
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Blue, Fe3O4, PLGA, etc.) for enhanced ultrasound imaging or high intensity focused ultrasound mediated therapy.[17] Thanks to the empty space in their hollow structure, the asprepared PEG-Bi2Se3 nanoparticles can also act as a carrier for loading of perfluorohexane (a kind of PFC) using the reported method.[17a,e] A large number of bubbles emerged in the PFCloaded hollow PEG-Bi2Se3 dispersion after it was heated over 65 °C, which was over the boiling point of perfluorohexane, suggesting the effective loading of PFC inside those nanoparticles (Figure S6, Supporting Information). Owing to the high solubility of oxygen in the PFC liquid, PFC-loaded hollow PEG-Bi2Se3 (PEG-Bi2Se3@PFC) may act as an oxygen reservoir, allowing gradual release of oxygen in an oxygen deficient environment (Figure 2a). In our experiments, hollow PEG-Bi2Se3 nanoparticles with PFC loading were saturated with oxygen (PEG-Bi2Se3@PFC@O2). The oxygen loading capacity of PEG-Bi2Se3@PFC was determined to be 96.9 ± 9.4 µmol per 1g of PEG-Bi2Se3 (Figure S7, Supporting Information). Once oxygen was loaded into the PEGBi2Se3@PFC nanoparticles, its retention in those nanoparticles could be more than 1 h (Figure S8, Supporting Information). In order to investigate the oxygen release, hollow PEG-Bi2Se3 nanoparticles with or without PFC loading were saturated with
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PEG-Bi2Se3@PFC@O2. Considering the fact that PEG-Bi2Se3 showed no obvious shape or size changes after NIR irradiation (Figure S10, Supporting Information), this burst release should be owing to the increase temperature triggered by NIR-induced photothermal effect. As a control, less increase of oxygen concentration in the PEG-Bi2Se3@O2 solution (oxygen saturated PEG-Bi2Se3) was observed under the same irradiation condition (Figure 3b). The oxygen concentrations decreased at the later stage of continuous NIR irradiation, simply because the PEGBi2Se3@PFC@O2 solution in this experiment was exposed to the nitrogen atmosphere, and thus the released oxygen from nanoparticles would be quickly diffused out. Notably, different from the solution sample tested here, a real tumor is a relatively closed system, and oxygen released from PEG-Bi2Se3@ PFC@O2 may probably stay in the tumor for a much longer period of time to allow the enhanced tumor oxygenation. Next, we conducted an in vivo tumor model animal experiment to verify the above hypothesis. Balb/c mice were obtained from Suzhou Belda Biopharmaceutical Co., Ltd and used under protocols approved by Soochow University Laboratory Animal Center. To demonstrate the photothermal effect of Bi2Se3 in vivo, the Balb/c mice, bearing subcutaneous 4T1 murine breast cancer tumors, were intratumorally injected with hollow PEG-Bi2Se3 (8 mg kg−1), and then irradiated with the 808 nm laser (0.5 W cm−2) for 10 min. As monitored by an IR thermal camera, during NIR irradiation, the temperatures of tumors injected with PEG-Bi2Se3 increased to ≈45 °C, while those injected with PBS were not notable heated (final temperature ≈35 °C) (Figure 3c,d). Therefore, PEG-Bi2Se3 could indeed serve as an effective photothermal agent to locally enhance the tumor temperature under exposure to the NIR laser. To confirm the ability of oxygen release from PEG-Bi2Se3@ PFC@O2 to overcome hypoxia in vivo, a hypoxia-probe (pimonidazole) immuno-histochemical assay was performed for 4T1 tumors after different treatments (see the method section in Supporting Information for details). Immunofluorescence staining assay was carried out by staining the cell nuclei and hypoxia areas with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (blue) and anti-pimonidazole antibody (green), respectively (Figure 3e,f). The 4T1 murine breast cancer tumors treated either with PEG-Bi2Se3+NIR or PEGBi2Se3@PFC@O2 showed weakened pimonidazole-stained (green) hypoxic signals compared with the control group, indicating that the tumor hypoxia could be partially reduced by the mild hyperthermia or O2 gradually released from PEG-Bi2Se3@ PFC@O2, respectively. More interestingly, the tumor treated with PEG-Bi2Se3@PFC@O2 + NIR showed the lowest hypoxic signals among all different groups, indicating the remarkably enhanced tumor oxygenation owing to the combined effect of the mild hyperthermia (Figure S11, Supporting Information) and the NIR-triggered burst release of O2 from PFC-loaded hollow nanoparticles. Finally, we would like to test whether the strategy of using NIR light to trigger burst release of oxygen so as to maximize the tumor oxygenation would be helpful to improve the efficacy of RT for in vivo cancer treatment. In our experiment, a total of seven groups of mice were used: Group 1: PBS; Group 2: PEGBi2Se3@PFC@O2 + NIR; Group 3: X-ray irradiation without nanoparticle injection (RT alone); Group 4: PEG-Bi2Se3 + RT;
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oxygen,[8b,9b] and then added into deoxygenated water in a flask filled with nitrogen. The dissolved oxygen concentrations in those solutions were monitored by a portable oxygen meter in real time (Figure 2b and Figure S9a, Supporting Information). Instantly after addition of these two samples, sharply increased oxygen concentrations were observed for both solutions (initial peak in those curves), and the one with addition of PEGBi2Se3@PFC@O2 showed higher oxygen concentration in the beginning. Thereafter, since the two solutions were placed in the nitrogen atmosphere, the dissolved oxygen concentrations should decrease overtime. Notably, the oxygen concentration in the solution added with PEG-Bi2Se3@PFC@O2 showed a much slower decrease compared to the solution with addition of oxygen saturated PEG-Bi2Se3, in which the dissolved oxygen concentration decreased rapidly within 20 min. Therefore, PEG-Bi2Se3@PFC is able to store more oxygen than PEGBi2Se3 alone, and could ensure gradual release of oxygen to the surrounding hypoxic environment. It has been reported that the increased oxygen level may result in greatly enhanced cellular damage induced by ionizing radiation.[1d,8g] In the meantime, Bi as a high-Z element can effectively concentrate a greater local radiation dose within the tumor, thus enhancing the RT efficacy to cancer.[5a,18] Next, we tested the capability of PEG-Bi2Se3@PFC@O2 to sensitize RT in vitro. 4T1 murine breast cancer cells were cultured at 1% O2, 5% CO2, and 94% N2 to mimic the hypoxic environment and then treated with hollow PEG-Bi2Se3, RT (6 Gy of X-ray irradiation), PEG-Bi2Se3 (100 µg mL−1) + RT (6 Gy), and PEG-Bi2Se3@ PFC@O2 (100 µg mL−1) + RT (6 Gy). The cells were then immunofluorescently labeled for γ-H2AX, a marker of double-strand DNA breaks, to evaluate the level of DNA damage in cancer cells induced by RT (Figure 2c,d). Cancer cells treated with PEG-Bi2Se3 + RT exhibited remarkably enhanced DNA damage than those treated with RT, and PEG-Bi2Se3 alone, suggesting the strong RT enhancement effect of those Bi-containing nanoparticles. More importantly, we observed that further enhanced DNA damage was induced by PEG-Bi2Se3@PFC@O2+RT, to a level even higher than that achieved by PEG-Bi2Se3+ RT, indicating the improved O2 supply released from PFC loaded inside those hollow nanoparticles could provide additional benefit for RT sensitization. When the RT treatment is conducted, the tumor is exposed to the X-ray beam at a desired energy for several minutes (≈5 min) in our experiments. In order to maximize the efficacy of RT, it would be essential to make sure the oxygen level in the tumor reached the maximal level within these few minutes during exposure to X-ray. Since Bi2Se3 has been found to be an effective photothermal agent to generate heat upon NIR laser exposure, we hypothesized that the increase temperature triggered by NIR light should naturally increase the diffusion/release of oxygen from PFC loaded inside our hollow nanoparticles. Therefore, after PEG-Bi2Se3@PFC@O2 was added into deoxygenated water, we used an NIR laser to irradiate the solution, whose oxygen concentration was recorded in real time. The temperature effect on the oxygen measurement was calibrated by a control experiment (Figure S9c, Supporting Information). Interestingly, when exposed to the laser, the PEG-Bi2Se3@PFC@O2 solution showed a rapid increase of oxygen concentration, indicating a burst release of oxygen from
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Figure 3. NIR-induced tumor oxygenation using oxygen-loaded nanoparticles. a) A scheme showing burst release of oxygen from PEG-Bi2Se3@PFC@ O2 under stimulation by an NIR laser. b) O2 concentration changes in solutions of PEG-Bi2Se3@O2 or PEG-Bi2Se3@PFC@O2 under irradiation by an 808 nm laser. c) IR thermal images of 4T1 tumor-bear mice with injection of PBS or PEG-Bi2Se3 (dose = 8 mg kg−1), under the 808 nm laser irradiation (0.5 W cm−2). d) Temperature changes of tumors monitored by the IR thermal camera during laser irradiation. e) Representative immunofluorescence images of tumor slices stained by the hypoxia-probe. The nuclei and hypoxia areas were stained with DAPI (blue), and anti-pimonidazole antibody (green), respectively. Dramatically enhanced oxygenation level was observed for tumors injected with PEG-Bi2Se3@PFC@O2 and exposed to the NIR light. f) Quantification of tumor hypoxia for different groups shown in (e). P values: *P < 0.05, **P < 0.01, ANOVA.
Group 5: PEG-Bi2Se3@PFC@O2 + RT; Group 6: PEG-Bi2Se3 + NIR + RT; Group 7: PEG-Bi2Se3@PFC@O2 + NIR + RT. The nanoparticle dose was 8 mg kg−1 in all groups. NIR irradiation was conducted by the 808 nm laser at 0.5 W cm−2 for 10 min, while the radiation dose used for RT was 6 Gy. The tumor growth curves and averaged final tumor weights of different groups of mice 16 d after various treatments are shown in Figure 4a,b, respectively. The representative photographs of mice in different groups are shown in Figure S12, Supporting Information. As expected, the mild photothermal treatment showed no significant effect to tumor growth (Group 2). In the meanwhile, RT alone by X-ray irradiation (Group 3) was only able to partially inhibit the tumor growth. Compared with RT alone (Group 3), PEG-Bi2Se3 + RT (Group 4) (P < 0.05) and PEG-Bi2Se3@PFC@O2 + RT (Group 5) (P < 0.05) induced notably enhanced tumor growth inhibition, and the efficacy achieved in Group 5 appeared to be slightly better than that of Group 4, illustrating that PEG-Bi2Se3 as an X-rayabsorbing agent could enhance the RT efficacy in vivo by locally
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concentrating radiation energy inside the tumor, and the gradually released oxygen from Bi2Se3@PFC@O2 could offer additional benefit to sensitize RT. Since mild PTT could promote the tumor oxygenation, PEG-Bi2Se3 + NIR + RT (Group 6) also resulted in a decent therapeutic outcome, similar to that achieved in Group 5 with oxygen-loaded nanoparticles. Most remarkably, it was found that PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) offered the most effective tumor growth inhibition compared to all the other groups (P < 0.05), demonstrating an enhanced synergistic therapeutic effect contributed from such NIR laser triggered O2 burst release to overcome tumor hypoxia. Pathology changes in tumors were further used to evaluate the RT efficacy. Two days after various treatments were conducted, the tumors from different groups were dissected for hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Micrographs of H&E and TUNELstained tumor slices (Figure 4c) uncovered that treatment by
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COMMUNICATION Figure 4. In vivo cancer treatment in a mouse tumor model. Seven groups of 4T1 tumor-bearing mice were treated with (1) PBS, (2) PEG-Bi2Se3@ PFC@O2 + NIR, (3) RT alone, (4) PEG-Bi2Se3 + RT, (5) PEG-Bi2Se3@PFC@O2 + RT, (6) PEG-Bi2Se3 + NIR + RT, and (7) PEG-Bi2Se3@PFC@O2 + NIR + RT. a) Tumor growth curves of different groups of mice after various treatments (5 mice for each group). b) Averaged weights of tumors collected from different groups of mice at day 16 post the initiation of treatments. NIR irradiation was conducted by the 808 nm laser at 0.5 W cm−2 for 10 min and the radiation dose used for RT was 6 Gy. P values: *P < 0.05, **P < 0.01, ANOVA. c) Microscopy images of H&E-stained and TUNEL-stained tumor slices collected from different groups of mice 2 d after treatments.
PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) exerted the most significant damages to tumor cells, to a level more severe compared to the other groups, although partial tumor cell damages and moderate levels of apoptosis were also noted in Groups 3–6. Such remarkable therapeutic effect by treating tumors with PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) could be attributed to the following multiple factors: i) Bi element in those nanoparticles is known to strongly absorb X-ray, and thus could enable the concentrated irradiation energy in the tumor to sensitize RT;[5a,18] ii) the mild photothermal effect induced by NIR laser could promote the blood flow into tumors and overcome tumor hypoxia particularly in areas with blood vessels;[5a,6a] iii) oxygen loaded inside those hollow nanoparticles could be rapidly released under NIR-triggered photothermal heating, so that the tumor oxygenation level may reach a peak level within a short period of time, during which RT is conducted to achieve the maximal therapeutic effect.
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To look at the potential side effect of those nanoparticles and such a treatment strategy, the main organs of mice treated with PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) were collected 16 d after treatment and sliced for H&E staining. From the histology examination, no noticeable tissue damage and adverse effect to major organs of animals was observed (Figure S13, Supporting Information). Meanwhile, the body weights of the mice were not significantly affected by various different treatments, indicating no acute side effects in this therapy delivered by PEG-Bi2Se3@PFC@O2 (Figure S14, Supporting Information). Despite the high levels of PEG-Bi2Se3 nanoparticles accumulated in the liver and spleen post intravenous injection (Figure S15, Supporting Information), no evidence of toxicity from the in vitro cellular cytotoxicity data (Figure S16, Supporting Information) and in vivo blood biochemistry/complete blood panel analysis results was found for PEG-Bi2Se3 within our tested dose range (Figure S17, Supporting Information). Those results suggested that our PEGylated hollow Bi2Se3
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nanoparticles may be a relatively safe agent, although more indepth toxicology evaluations are still needed in future studies. In our work, hollow Bi2Se3 nanoparticles are prepared by a facile cation exchange method via the nanoscale Kirkendall effect, which is featured with advantages such as the precise control of nanostructure sizes and shapes based on template nanoparticles. Those hollow Bi2Se3 nanoparticles are then functionalized with PEG, infused with PFC, and then loaded with oxygen. In such PEG-Bi2Se3@PFC@O2 system, Bi2Se3 could act as a radio-sensitizer by itself to enhance the efficacy of RT, while PFC loaded inside those hollow nanoparticles could be used as an oxygen carrier to moderately improve the tumor oxygenation. More remarkably, owing to the strong NIR absorbance of Bi2Se3, NIR laser irradiation could generate a strong photothermal effect, which then is able to trigger a burst release of oxygen to greatly promote the tumor oxygenation and further overcome the hypoxia-associated radio-resistance of tumors. As the result, a rather effective in vivo therapeutic outcome is achieved by treatment with NIR-enhanced RT using PEG-Bi2Se3@PFC@O2. Therefore, this work presents a novel nanotechnology approach to sensitize radiotherapy not only by concentrating radiation energy inside the tumor using a X-ray-absorbing agent, but also by improving tumor oxygenation to enhance the RT-induced tumor destruction, with the same agent carrying oxygen that can be released under an external optical stimulation. Moreover, the similar strategy/concept may be extended to other more clinically relevant settings, such as by using ultrasound to trigger the release of oxygen from O2-loaded PFC nano-droplets upon systemic administration, so as to enhance RT treatment of deeply located tumors that may not be easily accessed by light.
[3]
[4]
[5]
[6]
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
[7]
Acknowledgements This work was partially supported by the National Natural Science Foundation of China (51525203, 51302180, 51222203), the National “973” Program of China (2011CB911002, 2012CB932601), a Project Funded by Collaborative Innovation Center of Suzhou Nano Science & Technology, the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and Post-doctoral science foundation of China (2014M561706, 2013M531400). Note: The labeling in the caption of Figure 1 was corrected and Figure 3 was reset in color on April 13, 2016, after initial publication online.
[8]
Received: September 19, 2015 Revised: December 19, 2015 Published online: February 5, 2016
[1] a) P. Wardman, Clin. Oncol. UK 2007, 19, 397; b) A. C. Begg, F. A. Stewart, C. Vens, Nat. Rev. Cancer 2011, 11, 239; c) E. E. Deschner, L. H. Gray, Radiat. Res. 1959, 11, 115; d) B. J. Moeller, R. A. Richardson, M. W. Dewhirst, Cancer Metastasis Rev. 2007, 26, 241. [2] a) E. L. Jones, L. R. Prosnitz, M. W. Dewhirst, P. K. Marcom, P. H. Hardenbergh, L. B. Marks, D. M. Brizel, Z. Vujaskovic, Clin.
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Perfluorocarbon-Loaded Hollow Bi2Se3 Nanoparticles for Timely Supply of Oxygen under Near-Infrared Light to Enhance the Radiotherapy of Cancer Guosheng Song, Chao Liang, Xuan Yi, Qi Zhao, Liang Cheng, Kai Yang, and Zhuang Liu*
Radiation therapy (RT) is a widely used cancer treatment strategy, in which the ionizing radiation (e.g., X-ray, γ-ray) locally applied on the tumor is able to generate oxygen-centered radicals formed by the ionization of surrounding water to induce DNA damage and thereby kill cancer cells.[1] Especially, if oxygen is available, it can react with the broken ends of DNA to create stable organic peroxides, which cannot be easily repaired, greatly enhancing the degree of RT-induced cellular damage.[1a,c,d] However, during RT, normal cells under exposure to the radiation beam would also be nonselectively killed, resulting in severe toxic side effects.[2] On the other hand, since the degree of cellular damage caused by ionizing radiation is highly dependent on the level of cellular oxygenation,[1a] while the tumor microenvironment is more hypoxic than normal tissues, hypoxia-associated resistance of RT often occurs, particularly for cancer cells in the center of a tumor where the oxygen supply is deficient.[3] In recent years, various strategies including nanomedicine approaches have been explored to improve the therapeutic outcomes of RT in cancer treatment.[4] Many efforts have been devoted to the development of chemoradiotherapy by using nanoparticle-drugs to enhance the efficacy of RT.[5] It has also been found that tumor-homing nanoparticles containing heavy elements which exhibit strong X-ray attenuation ability may be used to sensitize RT by concentrating a great deal of irradiation energy inside the tumor, so as to enhance the efficacy and specificity of RT in cancer treatment.[5b,6] Moreover, several methods have been developed to overcome the hypoxia-associated RT resistance by challenging the hypoxic tumor microenvironment, such as the use of MnO2 nanoparticles, bioreductive pro-drugs (e.g., tirapazamine), as well as X-ray-controlled NOreleasing upconversion nanoparticles.[5c,7] In our recent work, it was found that the mild photothermal effect induced by nearinfrared (NIR)-light-absorbing nanoparticles could be utilized Dr. G. Song, C. Liang, Prof. L. Cheng, Prof. Z. Liu Institute of Functional Nano & Soft Materials (FUNSOM) Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices Soochow University Suzhou, Jiangsu 215123, China E-mail: [email protected] X. Yi, Q. Zhao, Prof. K. Yang School of Radiation Medicine and Protection & School for Radiological and Interdisciplinary Sciences (RAD-X) Medical College of Soochow University Suzhou, Jiangsu 215123, China
DOI: 10.1002/adma.201504617
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to increase intratumoral blood flow and subsequently improve the oxygenation status of tumor microenvironment, so as to enhance the therapeutic efficacy of RT.[5a] Perfluorocarbon (PFC) is widely used in ultrasound contrast imaging and its several gaseous formulations are commercially available for diagnostic imaging.[8] Moreover, liquid PFC can dissolve a large amount of oxygen and serve as an oxygen carrier, enabling it to be used as a blood substitute.[8a,b,9] The PFC emulsions (such as Fluosol-DA- 20%) have been approved for intravenous use by the United States Food and Drug Administration.[10] In this work, hollow Bi2Se3 nanoparticles, which are able to absorb both NIR light and X-ray irradiation, are prepared by a facile cation exchange method and then functionalized with polyethylene glycol (PEG). Interestingly, those nanoparticles with the hollow structure could be effectively filled with perfluorohexane (a kind of liquid PFC), which then is able to act as an oxygen reservoir. After being saturated with oxygen, the obtained PEG-Bi2Se3@PFC@O2 can gradually release oxygen and offer significantly increased radiation-induced DNA damage of cancer cells during RT, in comparison to that achieved with PEG-Bi2Se3 alone. Importantly, we further uncover that exposure of PEG-Bi2Se3@PFC@O2 to NIR light would result in the burst release of oxygen from nanoparticles and thus the instantly increased tumor oxygenation, which is greatly helpful to overcome the hypoxia-associated RT-resistance. Since Bi2Se3 by itself could also sensitize RT by concentrating irradiation energy in the tumor, RT treatment of cancer with PEG-Bi2Se3@ PFC@O2 under enhancement with NIR light is found to be extremely effective in preventing the tumor growth. In our previous report, a cation exchange method was used to produce MnSe@Bi2Se3 core-shell nanostructures from MnSe nanoparticles with a relatively large size (average length of ≈132 nm and width of ≈105 nm) at the temperature of 180 °C.[5a] It is hypothesized that if a smaller template with a high surface-to-volume ratio is used, the rate of ions outward diffusion should be dramatically increased than that of inward diffusion, resulting in a cavity within the nanoparticle due to the nanoscale Kirkendall effect.[11] Herein, smaller MnSe nanoparticles were used as templates, starting from which hollow Bi2Se3 nanoparticles were prepared by a facile one-pot cation exchange method and Kirkendall effect (Figure 1a). In brief, MnSe nanoparticles with the average diameter of ≈35 nm as templates were pre-made (Figure S1, Supporting Information)[12] and then added with bismuth source under heating. The higher pKsp value of bismuth chalcogenides than manganese chalcogenides would be favorable to promote cation exchange of manganese by bismuth.[13]
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COMMUNICATION Figure 1. Synthesis and characterization of hollow Bi2Se3 nanoparticles. a) A scheme showing the preparation of hollow Bi2Se3 nanoparticles using MnSe nanoparticles as the template. b) TEM images of core-shell MnSe@Bi2Se3 prepared at 140 °C (Inset: the corresponding magnified TEM image). c) HAADF-STEM image of core-shell MnSe@Bi2Se3 prepared at 140 °C. The element maps showed the distribution of Mn, Se, and Bi. d) TEM images of hollow Bi2Se3 nanoparticles prepared at 180 °C (Inset: the corresponding magnified TEM image). e) EDX spectrum of hollow Bi2Se3 prepared at 180 °C. No Mn signal was found in the EDX spectrum of those hollow nanostructures. f) UV–vis–NIR absorbance spectra of PEG-Bi2Se3 nanoparticles in aqueous dispersions with various concentrations. g) Temperature elevation curves of aqueous solutions containing PEG-Bi2Se3 with different concentrations, under the irradiation of an 808 nm laser (0.8 W cm−2).
We carefully evaluated how the heating temperature would affect the final nanoparticle structure. It has been found that when the reaction temperature reached 120 °C (Figure S2a, Supporting Information) and 140 °C (Figure 1b and Figure S2b, Supporting Information), Mn2+ would be partially exchanged by Bi3+ in the outer layer of nanoparticles, resulting in coreshell MnSe@Bi2Se3 nanoparticles as evidenced by transmission electron microscope (TEM) images (Figure 1b inset and Figure S2b inset, Supporting Information), the high-angle annular dark-field scanning TEM (HAADF-STEM)-based elemental mapping (Figure 1c), as well as X-ray diffraction (XRD) data (Figure S3, Supporting Information). With further increase of temperature from 160 °C (Figure S2c, Supporting Information) to 180 °C (Figure 1d and Figure S2d, Supporting Information), the core–shell nanostructure gradually transformed into the hollow Bi2Se3 structure. The resulted hollow Bi2Se3 nanoparticles showed a well-maintained morphology
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of MnSe templates with the uniform size of ≈35 nm and shell thickness of ≈8 nm. As expected, magnification of TEM images (Figure 1d inset and Figure S2b inset, Supporting Information), energy dispersive spectra (EDX) (Figure 1e), and XRD data (Figure S3, Supporting Information) further confirmed the successful fabrication of hollow Bi2Se3. Therefore, using small MnSe nanoparticles as the template, increase of temperature could firstly activate the cation exchange to form core–shell MnSe@Bi2Se3, and then promote the transformation of such core–shell nanoparticles into the hollow Bi2Se3 nanostructure, probably due to the outward diffusion of manganese ion accelerated by the nanoscale Kirkendall effect.[11,14] After transformation into hollow Bi2Se3, the specific surface area of those nanoparticles was measured to be 76.3 cm2 g−1 (Figure S4, Supporting Information), allowing the effectively loading of guest molecules in the inner cavity of such hollow nanostructure.
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Figure 2. Oxygen-loaded nanoparticles for enhanced radiotherapy. a) A scheme showing hollow PEG-Bi2Se3 nanoparticles with PFC loading as an oxygen carrier. b) O2 concentration changes after addition of PEG-Bi2Se3@O2 or PEG-Bi2Se3@PFC@O2 into deoxygenated water under a nitrogen atmosphere. c) γ-H2AX-stained 4T1 cells treated with PBS, PEG-Bi2Se3, RT, PEG-Bi2Se3 + RT, or PEG-Bi2Se3@PFC@O2 +RT, under a hypoxic condition. The nanoparticle concentration was 100 µg mL−1 and the irradiation dose was 6 Gy. Cells exposed to PEG-Bi2Se3@PFC@O2 nanoparticles after RT exhibited the highest level of DNA damage (positive in γ-H2AX). d) Quantitative analysis of γ-h2ax foci density (γ-h2ax foci/100 µm2) for n > 100 cells in each treatment group. P values: **P < 0.01, by analysis of variance (ANOVA).
A layer-by-layer (LBL) polymer coating method was then developed to modify those hollow Bi2Se3 nanoparticles,[15] whose surface was covered by oleylamine post-synthesis. Bi2Se3 nanoparticles were firstly modified with dimercaptosuccinic acid (DMSA), which would offer those nanoparticles negative charges and make them dispersible in water. Such DMSAmodified nanoparticles were then encapsulated with cationic poly(allylamine hydrochloride) (PAH) and anionic poly(acrylic acid) (PAA) in sequence. After further conjugation with amineterminated PEG (NH2-PEG, Mw = 5000), the resulted PEGylated hollow Bi2Se3 (PEG-Bi2Se3) showed great dispersity in various physiological solutions. The successful LBL coating was confirmed by the alternative change of zeta potentials (Figure S5a, Supporting Information) and gradual increase of hydrodynamic sizes (Figure S5b, Supporting Information) of those nanoparticles after different layers of polymers were coated. Moreover, PEG-Bi2Se3 possessed strong absorbance in NIR region (Figure 1f), and thus exhibited an outstanding photothermal effect upon irradiation by an 808 nm NIR laser (Figure 1g). PFC can dissolve a large amount of oxygen due to the van der Waals interaction between PFC and oxygen.[16] Previous studies have demonstrated that PFC could be infused into the cavity of various hollow nanostructures (hollow SiO2, hollow Prussian 2718
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Blue, Fe3O4, PLGA, etc.) for enhanced ultrasound imaging or high intensity focused ultrasound mediated therapy.[17] Thanks to the empty space in their hollow structure, the asprepared PEG-Bi2Se3 nanoparticles can also act as a carrier for loading of perfluorohexane (a kind of PFC) using the reported method.[17a,e] A large number of bubbles emerged in the PFCloaded hollow PEG-Bi2Se3 dispersion after it was heated over 65 °C, which was over the boiling point of perfluorohexane, suggesting the effective loading of PFC inside those nanoparticles (Figure S6, Supporting Information). Owing to the high solubility of oxygen in the PFC liquid, PFC-loaded hollow PEG-Bi2Se3 (PEG-Bi2Se3@PFC) may act as an oxygen reservoir, allowing gradual release of oxygen in an oxygen deficient environment (Figure 2a). In our experiments, hollow PEG-Bi2Se3 nanoparticles with PFC loading were saturated with oxygen (PEG-Bi2Se3@PFC@O2). The oxygen loading capacity of PEG-Bi2Se3@PFC was determined to be 96.9 ± 9.4 µmol per 1g of PEG-Bi2Se3 (Figure S7, Supporting Information). Once oxygen was loaded into the PEGBi2Se3@PFC nanoparticles, its retention in those nanoparticles could be more than 1 h (Figure S8, Supporting Information). In order to investigate the oxygen release, hollow PEG-Bi2Se3 nanoparticles with or without PFC loading were saturated with
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PEG-Bi2Se3@PFC@O2. Considering the fact that PEG-Bi2Se3 showed no obvious shape or size changes after NIR irradiation (Figure S10, Supporting Information), this burst release should be owing to the increase temperature triggered by NIR-induced photothermal effect. As a control, less increase of oxygen concentration in the PEG-Bi2Se3@O2 solution (oxygen saturated PEG-Bi2Se3) was observed under the same irradiation condition (Figure 3b). The oxygen concentrations decreased at the later stage of continuous NIR irradiation, simply because the PEGBi2Se3@PFC@O2 solution in this experiment was exposed to the nitrogen atmosphere, and thus the released oxygen from nanoparticles would be quickly diffused out. Notably, different from the solution sample tested here, a real tumor is a relatively closed system, and oxygen released from PEG-Bi2Se3@ PFC@O2 may probably stay in the tumor for a much longer period of time to allow the enhanced tumor oxygenation. Next, we conducted an in vivo tumor model animal experiment to verify the above hypothesis. Balb/c mice were obtained from Suzhou Belda Biopharmaceutical Co., Ltd and used under protocols approved by Soochow University Laboratory Animal Center. To demonstrate the photothermal effect of Bi2Se3 in vivo, the Balb/c mice, bearing subcutaneous 4T1 murine breast cancer tumors, were intratumorally injected with hollow PEG-Bi2Se3 (8 mg kg−1), and then irradiated with the 808 nm laser (0.5 W cm−2) for 10 min. As monitored by an IR thermal camera, during NIR irradiation, the temperatures of tumors injected with PEG-Bi2Se3 increased to ≈45 °C, while those injected with PBS were not notable heated (final temperature ≈35 °C) (Figure 3c,d). Therefore, PEG-Bi2Se3 could indeed serve as an effective photothermal agent to locally enhance the tumor temperature under exposure to the NIR laser. To confirm the ability of oxygen release from PEG-Bi2Se3@ PFC@O2 to overcome hypoxia in vivo, a hypoxia-probe (pimonidazole) immuno-histochemical assay was performed for 4T1 tumors after different treatments (see the method section in Supporting Information for details). Immunofluorescence staining assay was carried out by staining the cell nuclei and hypoxia areas with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (blue) and anti-pimonidazole antibody (green), respectively (Figure 3e,f). The 4T1 murine breast cancer tumors treated either with PEG-Bi2Se3+NIR or PEGBi2Se3@PFC@O2 showed weakened pimonidazole-stained (green) hypoxic signals compared with the control group, indicating that the tumor hypoxia could be partially reduced by the mild hyperthermia or O2 gradually released from PEG-Bi2Se3@ PFC@O2, respectively. More interestingly, the tumor treated with PEG-Bi2Se3@PFC@O2 + NIR showed the lowest hypoxic signals among all different groups, indicating the remarkably enhanced tumor oxygenation owing to the combined effect of the mild hyperthermia (Figure S11, Supporting Information) and the NIR-triggered burst release of O2 from PFC-loaded hollow nanoparticles. Finally, we would like to test whether the strategy of using NIR light to trigger burst release of oxygen so as to maximize the tumor oxygenation would be helpful to improve the efficacy of RT for in vivo cancer treatment. In our experiment, a total of seven groups of mice were used: Group 1: PBS; Group 2: PEGBi2Se3@PFC@O2 + NIR; Group 3: X-ray irradiation without nanoparticle injection (RT alone); Group 4: PEG-Bi2Se3 + RT;
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oxygen,[8b,9b] and then added into deoxygenated water in a flask filled with nitrogen. The dissolved oxygen concentrations in those solutions were monitored by a portable oxygen meter in real time (Figure 2b and Figure S9a, Supporting Information). Instantly after addition of these two samples, sharply increased oxygen concentrations were observed for both solutions (initial peak in those curves), and the one with addition of PEGBi2Se3@PFC@O2 showed higher oxygen concentration in the beginning. Thereafter, since the two solutions were placed in the nitrogen atmosphere, the dissolved oxygen concentrations should decrease overtime. Notably, the oxygen concentration in the solution added with PEG-Bi2Se3@PFC@O2 showed a much slower decrease compared to the solution with addition of oxygen saturated PEG-Bi2Se3, in which the dissolved oxygen concentration decreased rapidly within 20 min. Therefore, PEG-Bi2Se3@PFC is able to store more oxygen than PEGBi2Se3 alone, and could ensure gradual release of oxygen to the surrounding hypoxic environment. It has been reported that the increased oxygen level may result in greatly enhanced cellular damage induced by ionizing radiation.[1d,8g] In the meantime, Bi as a high-Z element can effectively concentrate a greater local radiation dose within the tumor, thus enhancing the RT efficacy to cancer.[5a,18] Next, we tested the capability of PEG-Bi2Se3@PFC@O2 to sensitize RT in vitro. 4T1 murine breast cancer cells were cultured at 1% O2, 5% CO2, and 94% N2 to mimic the hypoxic environment and then treated with hollow PEG-Bi2Se3, RT (6 Gy of X-ray irradiation), PEG-Bi2Se3 (100 µg mL−1) + RT (6 Gy), and PEG-Bi2Se3@ PFC@O2 (100 µg mL−1) + RT (6 Gy). The cells were then immunofluorescently labeled for γ-H2AX, a marker of double-strand DNA breaks, to evaluate the level of DNA damage in cancer cells induced by RT (Figure 2c,d). Cancer cells treated with PEG-Bi2Se3 + RT exhibited remarkably enhanced DNA damage than those treated with RT, and PEG-Bi2Se3 alone, suggesting the strong RT enhancement effect of those Bi-containing nanoparticles. More importantly, we observed that further enhanced DNA damage was induced by PEG-Bi2Se3@PFC@O2+RT, to a level even higher than that achieved by PEG-Bi2Se3+ RT, indicating the improved O2 supply released from PFC loaded inside those hollow nanoparticles could provide additional benefit for RT sensitization. When the RT treatment is conducted, the tumor is exposed to the X-ray beam at a desired energy for several minutes (≈5 min) in our experiments. In order to maximize the efficacy of RT, it would be essential to make sure the oxygen level in the tumor reached the maximal level within these few minutes during exposure to X-ray. Since Bi2Se3 has been found to be an effective photothermal agent to generate heat upon NIR laser exposure, we hypothesized that the increase temperature triggered by NIR light should naturally increase the diffusion/release of oxygen from PFC loaded inside our hollow nanoparticles. Therefore, after PEG-Bi2Se3@PFC@O2 was added into deoxygenated water, we used an NIR laser to irradiate the solution, whose oxygen concentration was recorded in real time. The temperature effect on the oxygen measurement was calibrated by a control experiment (Figure S9c, Supporting Information). Interestingly, when exposed to the laser, the PEG-Bi2Se3@PFC@O2 solution showed a rapid increase of oxygen concentration, indicating a burst release of oxygen from
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Figure 3. NIR-induced tumor oxygenation using oxygen-loaded nanoparticles. a) A scheme showing burst release of oxygen from PEG-Bi2Se3@PFC@ O2 under stimulation by an NIR laser. b) O2 concentration changes in solutions of PEG-Bi2Se3@O2 or PEG-Bi2Se3@PFC@O2 under irradiation by an 808 nm laser. c) IR thermal images of 4T1 tumor-bear mice with injection of PBS or PEG-Bi2Se3 (dose = 8 mg kg−1), under the 808 nm laser irradiation (0.5 W cm−2). d) Temperature changes of tumors monitored by the IR thermal camera during laser irradiation. e) Representative immunofluorescence images of tumor slices stained by the hypoxia-probe. The nuclei and hypoxia areas were stained with DAPI (blue), and anti-pimonidazole antibody (green), respectively. Dramatically enhanced oxygenation level was observed for tumors injected with PEG-Bi2Se3@PFC@O2 and exposed to the NIR light. f) Quantification of tumor hypoxia for different groups shown in (e). P values: *P < 0.05, **P < 0.01, ANOVA.
Group 5: PEG-Bi2Se3@PFC@O2 + RT; Group 6: PEG-Bi2Se3 + NIR + RT; Group 7: PEG-Bi2Se3@PFC@O2 + NIR + RT. The nanoparticle dose was 8 mg kg−1 in all groups. NIR irradiation was conducted by the 808 nm laser at 0.5 W cm−2 for 10 min, while the radiation dose used for RT was 6 Gy. The tumor growth curves and averaged final tumor weights of different groups of mice 16 d after various treatments are shown in Figure 4a,b, respectively. The representative photographs of mice in different groups are shown in Figure S12, Supporting Information. As expected, the mild photothermal treatment showed no significant effect to tumor growth (Group 2). In the meanwhile, RT alone by X-ray irradiation (Group 3) was only able to partially inhibit the tumor growth. Compared with RT alone (Group 3), PEG-Bi2Se3 + RT (Group 4) (P < 0.05) and PEG-Bi2Se3@PFC@O2 + RT (Group 5) (P < 0.05) induced notably enhanced tumor growth inhibition, and the efficacy achieved in Group 5 appeared to be slightly better than that of Group 4, illustrating that PEG-Bi2Se3 as an X-rayabsorbing agent could enhance the RT efficacy in vivo by locally
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concentrating radiation energy inside the tumor, and the gradually released oxygen from Bi2Se3@PFC@O2 could offer additional benefit to sensitize RT. Since mild PTT could promote the tumor oxygenation, PEG-Bi2Se3 + NIR + RT (Group 6) also resulted in a decent therapeutic outcome, similar to that achieved in Group 5 with oxygen-loaded nanoparticles. Most remarkably, it was found that PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) offered the most effective tumor growth inhibition compared to all the other groups (P < 0.05), demonstrating an enhanced synergistic therapeutic effect contributed from such NIR laser triggered O2 burst release to overcome tumor hypoxia. Pathology changes in tumors were further used to evaluate the RT efficacy. Two days after various treatments were conducted, the tumors from different groups were dissected for hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Micrographs of H&E and TUNELstained tumor slices (Figure 4c) uncovered that treatment by
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COMMUNICATION Figure 4. In vivo cancer treatment in a mouse tumor model. Seven groups of 4T1 tumor-bearing mice were treated with (1) PBS, (2) PEG-Bi2Se3@ PFC@O2 + NIR, (3) RT alone, (4) PEG-Bi2Se3 + RT, (5) PEG-Bi2Se3@PFC@O2 + RT, (6) PEG-Bi2Se3 + NIR + RT, and (7) PEG-Bi2Se3@PFC@O2 + NIR + RT. a) Tumor growth curves of different groups of mice after various treatments (5 mice for each group). b) Averaged weights of tumors collected from different groups of mice at day 16 post the initiation of treatments. NIR irradiation was conducted by the 808 nm laser at 0.5 W cm−2 for 10 min and the radiation dose used for RT was 6 Gy. P values: *P < 0.05, **P < 0.01, ANOVA. c) Microscopy images of H&E-stained and TUNEL-stained tumor slices collected from different groups of mice 2 d after treatments.
PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) exerted the most significant damages to tumor cells, to a level more severe compared to the other groups, although partial tumor cell damages and moderate levels of apoptosis were also noted in Groups 3–6. Such remarkable therapeutic effect by treating tumors with PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) could be attributed to the following multiple factors: i) Bi element in those nanoparticles is known to strongly absorb X-ray, and thus could enable the concentrated irradiation energy in the tumor to sensitize RT;[5a,18] ii) the mild photothermal effect induced by NIR laser could promote the blood flow into tumors and overcome tumor hypoxia particularly in areas with blood vessels;[5a,6a] iii) oxygen loaded inside those hollow nanoparticles could be rapidly released under NIR-triggered photothermal heating, so that the tumor oxygenation level may reach a peak level within a short period of time, during which RT is conducted to achieve the maximal therapeutic effect.
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To look at the potential side effect of those nanoparticles and such a treatment strategy, the main organs of mice treated with PEG-Bi2Se3@PFC@O2 + NIR + RT (Group 7) were collected 16 d after treatment and sliced for H&E staining. From the histology examination, no noticeable tissue damage and adverse effect to major organs of animals was observed (Figure S13, Supporting Information). Meanwhile, the body weights of the mice were not significantly affected by various different treatments, indicating no acute side effects in this therapy delivered by PEG-Bi2Se3@PFC@O2 (Figure S14, Supporting Information). Despite the high levels of PEG-Bi2Se3 nanoparticles accumulated in the liver and spleen post intravenous injection (Figure S15, Supporting Information), no evidence of toxicity from the in vitro cellular cytotoxicity data (Figure S16, Supporting Information) and in vivo blood biochemistry/complete blood panel analysis results was found for PEG-Bi2Se3 within our tested dose range (Figure S17, Supporting Information). Those results suggested that our PEGylated hollow Bi2Se3
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nanoparticles may be a relatively safe agent, although more indepth toxicology evaluations are still needed in future studies. In our work, hollow Bi2Se3 nanoparticles are prepared by a facile cation exchange method via the nanoscale Kirkendall effect, which is featured with advantages such as the precise control of nanostructure sizes and shapes based on template nanoparticles. Those hollow Bi2Se3 nanoparticles are then functionalized with PEG, infused with PFC, and then loaded with oxygen. In such PEG-Bi2Se3@PFC@O2 system, Bi2Se3 could act as a radio-sensitizer by itself to enhance the efficacy of RT, while PFC loaded inside those hollow nanoparticles could be used as an oxygen carrier to moderately improve the tumor oxygenation. More remarkably, owing to the strong NIR absorbance of Bi2Se3, NIR laser irradiation could generate a strong photothermal effect, which then is able to trigger a burst release of oxygen to greatly promote the tumor oxygenation and further overcome the hypoxia-associated radio-resistance of tumors. As the result, a rather effective in vivo therapeutic outcome is achieved by treatment with NIR-enhanced RT using PEG-Bi2Se3@PFC@O2. Therefore, this work presents a novel nanotechnology approach to sensitize radiotherapy not only by concentrating radiation energy inside the tumor using a X-ray-absorbing agent, but also by improving tumor oxygenation to enhance the RT-induced tumor destruction, with the same agent carrying oxygen that can be released under an external optical stimulation. Moreover, the similar strategy/concept may be extended to other more clinically relevant settings, such as by using ultrasound to trigger the release of oxygen from O2-loaded PFC nano-droplets upon systemic administration, so as to enhance RT treatment of deeply located tumors that may not be easily accessed by light.
[3]
[4]
[5]
[6]
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
[7]
Acknowledgements This work was partially supported by the National Natural Science Foundation of China (51525203, 51302180, 51222203), the National “973” Program of China (2011CB911002, 2012CB932601), a Project Funded by Collaborative Innovation Center of Suzhou Nano Science & Technology, the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and Post-doctoral science foundation of China (2014M561706, 2013M531400). Note: The labeling in the caption of Figure 1 was corrected and Figure 3 was reset in color on April 13, 2016, after initial publication online.
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Received: September 19, 2015 Revised: December 19, 2015 Published online: February 5, 2016
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