A Near Infrared Light Triggered Hydrogenated Black TiO2 for Cancer Photothermal Therapy.

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A Near Infrared Light Triggered Hydrogenated Black TiO2 for Cancer Photothermal Therapy Wenzhi Ren, Yong Yan, Leyong Zeng, Zhenzhi Shi, An Gong, Peter Schaaf, Dong Wang,* Jinshun Zhao, Baobo Zou, Hongsheng Yu, Ge Chen,* Eric Michael Bratsolias Brown, and Aiguo Wu* delivery, and even TiO2 nanotube for photothermal therapy (PTT).[2] According to the reports, as an inorganic photosensitizer for cancer PDT is the most widely and important application of TiO2 nanoparticles (NPs) in biomedicine.[3] The principle of PDT is based on the accumulation of a photosensitizer in a tumor; light of a specified wavelength is applied resulting in the generation of reactive oxygen species (ROS) and subsequent killing of tumor cells. The band gap of TiO2 is 3.23 eV for anatase, which is equal to the energy of UV light at 385 nm. Therefore, upon irradiating light with energies higher than 385 nm (i.e., lower wavelengths), the photoinduced pairs of electrons and holes can induce ROS including O2− and OH• radicals leading to the killing of cancer cells.[4] Based on the theory of UV light–triggered radical production, cancer PDT of TiO2 NPs have attracted much attention over the last two decades. However, due to very shallow penetration and toxicity of UV light, cancer PDT with TiO2 NPs met obstacles that impeded further clinical applications. Many researchers attempted to change the exciting light from UV to visible light through doping or surface modification, although these advances solved the problem in some extent.[5] Visible light

White TiO2 nanoparticles (NPs) have been widely used for cancer photodynamic therapy based on their ultraviolet light–triggered properties. To date, biomedical applications using white TiO2 NPs have been limited, since ultraviolet light is a well-known mutagen and shallow penetration. This work is the first report about hydrogenated black TiO2 (H-TiO2) NPs with near infrared absorption explored as photothermal agent for cancer photothermal therapy to circumvent the obstacle of ultraviolet light excitation. Here, it is shown that photothermal effect of H-TiO2 NPs can be attributed to their dramatically enhanced nonradiative recombination. After polyethylene glycol (PEG) coating, H-TiO2-PEG NPs exhibit high photothermal conversion efficiency of 40.8%, and stable size distribution in serum solution. The toxicity and cancer therapy effect of H-TiO2-PEG NPs are relative systemically evaluated in vitro and in vivo. The findings herein demonstrate that infrared-irradiated H-TiO2PEG NPs exhibit low toxicity, high efficiency as a photothermal agent for cancer therapy, and are promising for further biomedical applications.

1. Introduction TiO2 nanomaterials have been widely used in many fields such as energy, environment, cosmetics, food, and biomedicine.[1] Its low toxicity, good biocompatibility, stable structure, and unique photocatalytic properties, make TiO2 nanomaterials be applied in cancer therapy, such as photodynamic therapy (PDT), drug

W. Ren, Dr. L. Zeng, Dr. Z. Shi, A. Gong, Prof. A. Wu Key Laboratory of Magnetic Materials and Devices & Division of Functional Materials and Nanodevices Ningbo Institute of Materials Technology and Engineering Chinese Academy of Sciences 1219 ZhongGuan West Road, Ningbo 315201, China E-mail: [email protected] Y. Yan, Prof. P. Schaaf, Dr. D. Wang Chair Materials for Electrical Engineering and Electronics Institute of Materials Engineering and Institute of Micro- and Nanotechnologies MarcoNano, TU Ilmenau Gustav-Kirchhoff-Str. 5, Ilmenau 98693, Germany E-mail: [email protected] Prof. J. Zhao, Prof. B. Zou Public Health Department Ningbo University 818 Fenghua Road, Ningbo 315211, China

Prof. H. Yu Affiliated Hospital of Medical School Ningbo University 247 People Road, Ningbo 315020, China Prof. G. Chen College of Environmental & Energy Engineering Beijing University of Technology 100 Pingleyuan, Beijing 100124, China E-mail: [email protected] Prof. E. M. B. Brown Department of Biological Sciences University of Wisconsin-Whitewater 800 W. Main St., Whitewater, WI 53190, USA

DOI: 10.1002/adhm.201500273

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that the facile synthetic H-TiO2 NPs are promising for further applications in biomedicine.

2. Results and Discussion 2.1. Photothermal Principle of H-TiO2 NPs

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activation does circumvent the mutagenic problems of UV excitation, but it still does not provide optimal penetration through biological tissue compared with near infrared (NIR) light. In 2011, Chen et al. reported that hydrogenated black TiO2 (H-TiO2) NPs with enhanced visible and NIR light absorption exhibited substantial solar-driven photocatalytic activities, including the photo-oxidation of organic molecules in water and the generation of hydrogen through photocatalytic water splitting.[6] H-TiO2 then attracted wide research attention in applications of sustainable energy sources and cleaning of the environment.[7] Although it has been demonstrated that the enhanced visible and NIR light absorption of H-TiO2 NPs was mainly due to the formation of oxygen vacancies in the H-TiO2 NPs, but the detailed mechanism for the enhanced photocatalytic performance of H-TiO2 NPs is still unknown and under debate.[8] However, it is accepted that H-TiO2 NPs possess strong NIR absorption, which is a desired attribute of any agents for cancer PTT. As an effective treatment for cancer, the principle of PTT is based on accumulation of photothermal agents in tumor which absorbs and converts NIR light into heat to kill the cancer cells. Compared with traditional therapeutic ways of cancer, such as chemotherapy, radiotherapy, and surgery, PTT is targeted, noninvasive, and consequently highly effective, but it does not involve the side effects of traditional therapies. The type of photothermal agent is a key factor for PTT and has attracted numerous research attentions.[9] Recently, a variety of inorganic and organic nanomaterials with high photothermal performance have been explored as effective photothermal agents for cancer therapy.[10] Based on the dramatic NIR absorption of H-TiO2 NPs, there is much promise to develop a new NIR triggered photothermal agent; however, there is still no report of H-TiO2 applied in biomedicine, especially in diagnosis and therapy of cancer.[11] Herein, we first attempt to study in hydrogenated black TiO2 NPs even hydrogenated semiconducting nanomaterials applied in the field of cancer diagnosis and therapy. In this work, H-TiO2 NPs were coated with polyethylene glycol (PEG) to improve the stability in physiological environment. Photothermal effect of PEG-coated H-TiO2 (H-TiO2-PEG) was measured. Finally, the toxicity and therapy effects of H-TiO2-PEG NPs were relative systemically evaluated in vitro and in vivo. Our results demonstrated that H-TiO2-PEG NPs possess 40.8% of photothermal conversion efficiency, low toxicity, and high anticancer effect in vitro and in vivo. These findings suggest

H-TiO2 NPs were obtained by a high-power density H2 plasma treatment based on our previous studies.[7b,c] Figure 1a shows the UV–vis–NIR absorption spectra of pristine- and H-TiO2 NPs. The highly increased absorption of H-TiO2 NPs in the region of visible and NIR light clarified their color change from white to black (as seen in the insets in Figure 1a), which might be correlated with a large amount of deep level defects (Ti3+ species) after H2 plasma treatment.[12] In addition, electron paramagnetic resonance (EPR) was measured to investigate the concentration of defects in H-TiO2 NPs. As indicated in Figure 1b, no obvious signal was observed for pristine-TiO2 NPs, indicating their limited amount of defects; while the H-TiO2 NPs showed a much stronger signal at an average g value of ≈1.957, implying the presence of a large amount of Ti3+ species in the bulk of the NPs.[13] It has been demonstrated that Ti3+ species created by hydrogenation process could induce the formation of additional electronic states below the conduction band of TiO2. In this case, H-TiO2 NPs with the substantial enhancement of visible and near infrared light absorption might be attributed to the transitions from the TiO2 valence band to these additional electronic states or from these additional electronic states to the TiO2 conduction band.[14] To understand the photothermal effect of H-TiO2 NPs through studying the properties of photogenerated charges of H-TiO2, light-induced EPR measurements were performed under 405 nm light irradiation (Figure 1c). For comparison, these spectra of pristine-TiO2 and H-TiO2 NPs have been subtracted by that of the spectra without light irradiation. The pristine-TiO2 NPs showed two well-separated sets of resonance lines. The high-field small peaks with an average g value of ≈1.960 can be assigned to electrons trapped on Ti3+ centers; the low-field sharp features with an average g value of ≈2.013, correspond to the holes trapped on O− sites.[15] These results indicate that the separation of photogenerated electrons and holes was more efficient in pristine-TiO2 than in H-TiO2 NPs. On the other hand, the intensity of O− signals was significantly decreased for H-TiO2 NPs, and an inverted broad resonance

Figure 1. a) UV–vis–NIR absorption spectra, b) EPR spectra, and c) light-induced EPR spectra of the pristine- and H-TiO2 NPs. The inserts show the pristine- and H-TiO2 NPs samples.




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was observed at an average g value of ≈1.957. This implies that decreasing the amount of Ti3+ species in the light-irradiated H-TiO2 may derive from the combination of the photogenerated holes and the localized bulk Ti3+ species. These results suggest that bulk Ti3+ species tend to act as charge carrier traps where most of the photogenerated holes were consumed through recombination with electrons.[8,16] According to the literature, nonradiative recombination releasing phonons is a major pathway for the annihilation of photogenerated charges in the semiconductor with high concentration of deep level defects, and the energy is exchanged in the form of lattice vibration and the thermal energy in materials is increased in this process.[17] Hence, the photothermal effect of H-TiO2 NPs can be attributed to their dramatically enhanced nonradiative recombination by deep level defects (Ti3+ species) which have also been observed in our previous work.[7]

2.2. Photothermal Conversion Efficiency of H-TiO2 NPs Based on the dramatic absorption of NIR and clarified principle of photothermal effect, H-TiO2 NPs were used as photothermal agent in the following research. To enhance the stability of H-TiO2 NPs in aqueous solutions, the NPs were coated by PEG to form H-TiO2-PEG NPs. As shown in Figure S1 (Supporting Information), due to H-TiO2 NPs were hydrogenated from Degussa P25 (commercial TiO2 NPs), size of H-TiO2 NPs is in accordance with P25, and is about 25 nm. Although H-TiO2-PEG NPs show a little aggregation in some extent, but they still show better dispersion ability than H-TiO2 NPs under the same measurement concentration (10 µg mL−1), which indicates PEG coating can increase dispersion of H-TiO2 NPs. Figure S2 (Supporting Information) shows hydrodynamic size distributions of H-TiO2 NPs before and after PEG coating. Average sizes of H-TiO2 and H-TiO2-PEG NPs are about 3705 and 205 nm, respectively, which also suggests PEG coating significantly improves dispersion of the NPs in water. As shown in Figure S3 (Supporting Information), zeta potentials of H-TiO2 NPs and H-TiO2-PEG NPs are −13.40 ± 3.88 and −15.00 ± 4.46 mV, respectively, and are not significantly different. For further

evaluation of the stability of H-TiO2-PEG NPs, the NPs were dispersed in serum solution for 7 d at room temperature. As shown in Figure S4 (Supporting Information), the size distributions of the NPs do not show dramatic change during the period, The PDI value is 0.17 ± 0.03, which suggests H-TiO2PEG NPs are stable in serum solution, and can be used as a potential photothermal therapy (PTT) agent for further study. Figure S5 (Supporting Information) shows UV-vis-NIR absorption of H-TiO2-PEG NPs in aqueous solution. The absorbance is 0.49 at 808nm which is a basic datum in calculation of photothermal conversion. As a photothermal agent in PTT, photothermal conversion is a very important attribute. Consequently, the photothermal conversion performance of H-TiO2-PEG NPs was evaluated. H-TiO2-PEG was dispersed in water, and then irradiated with an 808 nm NIR laser at 2 W cm−2. Pure water was used as a negative control. As shown in Figure 2a, the temperature of H-TiO2PEG NPs increases rapidly under NIR irradiation. After irradiation for 600 s, the temperature of H-TiO2-PEG NPs aqueous dispersions is 66 °C and there is a temperature increase of 44 °C. By comparison, the temperature of pure water is 28.5 °C and the increase is only 4.5 °C. It has been demonstrated that the cancer cells can be easily killed by exposure to temperatures over 50 °C for few minutes,[10] and therefore H-TiO2-PEG NPs may be considered as a viable agent for photothermal therapy of cancer. After 600 s of irradiation, the NIR laser was shut off, and the decreased temperature was recorded for another 1140 s. The temperature change (ΔT) response to the NIR laser over a period of 1740 s is shown in Figure 2b. Linear time data versus −ln(θ) obtained from the cooling period of the NIR laser is shown in Figure S6 (Supporting Information). The photothermal conversion efficiency (η) of H-TiO2-PEG can be calculated according to the following equation[18]

η=

hA( ΔTmax,mix − ΔTmax,H2O ) I(1 − 10 − Aλ )

(1)

where h is the heat transfer coefficient, A is the surface area of the container, ΔTmax ,mix and ΔTmax ,H2O are the temperature change of the H-TiO2-PEG NPs dispersion and solvent (water) at the maximum steady-state temperature, respectively, I is the laser

Figure 2. a) Temperature evaluation of H-TiO2-PEG NPs (100 µg mL−1) and pure water with 808 nm laser irradiation at 2 W cm−2 for different times. b) The temperature change (ΔT) response to NIR laser on and off in period of 2100 s.

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FULL PAPER Figure 3. a) Cell viability of MCF-7 and 4T1 cells after incubation with increased dose of H-TiO2-PEG NPs for 24 h. Data are expressed as the mean ± standard (n = 5). b) Uptake of red fluorescence dye (ARS) stained TiO2-PEG NPs in MCF-7 and 4T1 cells. X-axis shows intensity of red fluorescence which indicates amount of the NPs absorbed by the cells. Each datum was obtained from 20 000 cells.

power, and Aλ is the absorbance of H-TiO2-PEG NPs at 808 nm. Details of the calculation are given in the Supporting Information. According to the equation, η value of H-TiO2-PEG was calculated to be about 40.8%.

2.3. Cytotoxicity and Uptake of H-TiO2 NPs In Vitro It has been demonstrated that bare, white TiO2 NPs are environmentally friendly and low toxicity.[1] However, there is still no report about the toxicity of H-TiO2 NPs. Thus, cytotoxicity of H-TiO2-PEG NPs on both human and murine breast cancer cells was carried out through the MTT method. As shown in Figure 3a, MCF-7 or 4T1 cells were incubated with 50–300 µg mL−1 of H-TiO2-PEG NPs for 24 h. The relative viability of the cancer cell is not decreased significantly, which suggests H-TiO2-PEG NPs are relatively low toxic in vitro. Previous report showed that TiO2 NPs can be stained by fluorescent dye (alizarin red S, ARS).[19] To investigate the uptake of H-TiO2-PEG in cancer cells, both MCF-7 and 4T1 cells were incubated with ARS-stained H-TiO2-PEG (H-TiO2-PEG-ARS). Free ARS incubated cells were used as negative control. The cells were collected and 20 000 cells were analyzed by flow cytometer in each group. As shown in Figure 3b, X-axis is red fluorescence signal of ARS. The cells incubated with free ARS show very weak fluorescence, and mean signal intensities are 3.20 (MCF-7) and 2.37 (4T1). However, cells incubated with H-TiO2-PEG-ARS show relative stronger signal, and mean fluorescence intensities are 13.80 (MCF-7) and 12.00 (4T1), respectively. The results indicate that H-TiO2-PEG NPs can be absorbed by both MCF-7 and 4T1 cells. These results further suggest H-TiO2-PEG NPs are relatively low toxic in vitro.

2.4. MTT and Calcein Acetoxymethyl Ester (AM)/Propidium Iodide (PI) Staining Assay of Photothermal Therapy In Vitro Based on the high photothermal conversion efficiency, stability in serum solution, and low toxicity, the photothermal therapeutic efficacy of H-TiO2-PEG NPs was evaluated on


cancer cells in vitro. MCF-7 and 4T1 cells were incubated with H-TiO2-PEG NPs, and were then irradiated with an 808 nm NIR laser for 0–5 min. Cells incubated without H-TiO2-PEG NPs were also irradiated by laser as a negative control. As shown in Figure 4a,b, neither control group of MCF-7 or 4T1 cells receiving only laser treatment (without using H-TiO2PEG NPs), shows any significant loss in viability after 5 min of irradiation. However, the viability of the cells incubated with H-TiO2-PEG NPs decreases significantly with increasing irradiation time, and more than 80% of both MCF-7 and 4T1 cells were killed upon 5 min of irradiation. These results demonstrate that H-TiO2-PEG NPs are effective PTT agent for cancer therapy in vitro. In addition, to further verify the PTT performance of H-TiO2PEG in vitro, the cells were stained with calcein AM and PI solutions, which can discern live or dead cells through emitted green or red fluorescence, respectively. As shown in Figure 4c, the majority of MCF-7 and 4T1 cells in control, NPs, and laser groups are alive (green). However, most of the cells are dead (red) in laser + NPs group, indicating that the H-TiO2-PEG NPs can be applied as an effective PTT agent in vitro and may be useful for in vivo applications as well.

2.5. Toxicity and Distribution In Vivo As a potential in vivo PTT agent, the toxicity of H-TiO2-PEG NPs must be evaluated in vivo. In this study, histological analysis and blood analysis were used to evaluate the toxicity of the NPs in vivo according to previous report.[20] Healthy Blab/c mice were injected with different doses of H-TiO2-PEG NPs, and saline injected mice were used as control. Over one month period, behaviors of mice such as eating, drinking, excretion, activity, and neurological status were observed. There is no significant difference in the above behaviors between control and H-TiO2-PEG-injected groups. After one month, the mice were sacrificed; the main organs and blood were analyzed. Figure 5a shows histological analyses of the organs including heart, liver, spleen, kidney, and lung. There is no detectable tissue damage or other lesions such as necrosis, inflammatory, or pulmonary



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Figure 4. Viability of a) MCF-7 and b) 4T1 cells treated with or without 100 µg mL−1 H-TiO2-PEG NPs and 808 nm laser irradiation at 2 W cm−2 for ≈5 min. Data are expressed as the mean ± standard (n = 5). Statistically significant differences were evaluated using the Student’s t-test (*p < 0.05, **p < 0.01, ns > 0.05). c) Microscope images of calcein AM (green, live cells) and propidium iodide (red, dead cells) costained MCF-7 or 4T1 cells treated with or without 100 µg mL−1 H-TiO2-PEG NPs and laser irradiation for 5 min. (Scale bar = 50 µm.)

fibrosis when comparing the H-TiO2-PEG-injected groups with control group. Figure 5b shows hematological analysis of the mice including white blood cell (WBC), red blood cell (RBC), platelet (PLT), and their relevant data such as neutrophil (NE), lymphocyte (LY), monocyte (MO), eosinophil (EO), basophil (BASO), and hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCHC), red blood cell distribution width (RDW-CV), platelet distribution width (PDW-CV), and mean platelet volume (MPV). Number and distribution changes of blood cell are an important indicator of disease. As shown in Figure 5b, there is no significant difference between control and H-TiO2-PEG-injected groups, suggesting the mice are healthy. Furthermore, blood biochemical analysis was carried out by blood autoanalyzer. Six important hepatic indicators for liver functions (direct bilirubin, DBIL; albumin, ALB; globin, GLOB; alkaline phosphatase, ALP; gamma glutamyl transpeptidase, GGT), three indicators for kidney functions (urea nitrogen, UREA; creatinine, CREA; uric acid, URCA), total cholesterol (CHOL), triglyceride (TG), and glucose (GLU) were evaluated. As shown in Figure 5c, H-TiO2PEG NPs injection does not cause significant change of these indicators compared with the control group. Our relative sys-

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temic results demonstrate that H-TiO2-PEG NPs are not toxic to the mice at the injected doses for one month. However, more efforts are still required to systematically evaluate the potential long-term toxicity of H-TiO2-PEG NPs at higher doses in vivo. In order to evaluate the distribution of H-TiO2-PEG NPS in tumor-bearing mice, contents of Ti in main organs were measured. As shown in Figure S7 (Supporting Information), after intravenously injected with H-TiO2-PEG for 24 h, the NPs accumulation in tumor is 9.43 ± 0.03 µg g−1. The distribution of H-TiO2-PEG in tumor-bearing mice is similar to other reported nanomaterials.[18]

2.6. Photothermal Therapy In Vivo Because H-TiO2-PEG NPs possess low toxicity and good biocompatibility in vitro and in vivo, and also show effective photothermal therapy in vitro, their application for cancer photothermal therapy in vivo was carried out in tumor-bearing mice. The mice injected with H-TiO2-PEG NPs and irradiated with NIR laser are named as laser + NPs group. Mice neither injected nor irradiated are control group. Mice injected with




FULL PAPER Figure 5. a) Histological analyses of mice main organs injected with saline or various doses of H-TiO2-PEG NPs. (Scale bar = 20 µm.) b) Hematological analysis and c) blood biochemical analysis of the mice. Data are expressed as the mean ± standard (n = 3). Statistically significant differences were evaluated using the Student’s t-test (*p < 0.05, **p < 0.01, ns > 0.05).

saline and irradiated with NIR laser are called laser group. Mice only injected with H-TiO2-PEG NPs are NPs group. In order to record temperature change of NIR irradiated tumor, photothermal imaging of tumor-bearing mice was measured. As shown in Figure 6a, temperatures of tumor site in laser and laser + NPs groups are imaged in each minute during NIR irradiation. In laser group, temperature of tumor site increases very slowly and limits after 5 min irradiation of 2 W cm−2 NIR and temperature is about 36.5 °C which is accepted and tolerated by tissue. However, the temperature increases quickly, and reaches about 52.6 °C under the same NIR irradiation in laser + NPs group. Whole body temperature imaging is shown in Figure 6b. Figure 6c shows the temperature change (ΔT) of tumor sites during NIR irradiation. ΔT is about 11 °C in laser group; however, the value is about 27.1 °C in laser + NPs group


which is 2.5 times more than the laser group. It has been considered that cancer cells can be easily killed in few minutes when their temperature is over 50 °C.[9] Therefore, the tumor could be ablated in laser + NPs group. In order to justify our supposition of tumor ablation, four mice were sacrificed immediately after NIR irradiation, and tumors were analyzed by hematoxylin and eosin (H&E) stain. As shown in Figure 7, there is no obvious pathological change in control, laser, and NPs groups. However, there are significant necrosis features in laser + NPs group, such as pyknosis, karyorrhexis, and karyolysis happening in nucleus region; cell membrane is destroyed and fused with intercellular substance to form a fuzzy red-stained substance without any granular structure. Moreover, red blood cells are observed (shown by green arrows) which indicates tumor vessels are also destroyed by



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Figure 6. a) Photothermal images of tumor site in Laser group (injected with saline) and Laser + NPs group (injected with NPs) during 5 min irradiation of 2 W cm−2 NIR. b) Whole body temperature images of the mice at the fifth min of NIR irradiation. c) Temperature change (ΔT) of tumor sites during NIR irradiation.

heat. These results justify our supposition that tumor cells can be ablated after H-TiO2-PEG NPs injection plus NIR irradiation. The changes of tumor volume were recorded in the following two weeks. As shown in Figure 8a,d, the tumors grow

consistently in control, laser, and NPs groups. The relative tumor volumes (V/VO) on 14th day are 12.30 ± 2.38 in the control group, 14.20 ± 3.45 in the laser group, and 13.71 ± 2.82 in the NPs group. These results demonstrate that laser

Figure 7. Histological HE stain analysis of tumor injury after the tumor-bearing mice injected with or without H-TiO2-PEG NPs, and irradiated with or without 808 nm NIR for 5 min.

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FULL PAPER Figure 8. a) The relative tumor volume, b) body weight, and c) survival rate of the mice after different treatments described above. Data are expressed as the mean ± standard (n = 5). Statistically significant differences were evaluated using the Student's t-test (*p < 0.05, **p < 0.01, ns > 0.05). d) Photos of 4T1 tumor-bearing mice at the 1st, 5th, and 14th day after the treatments.

irradiation alone or H-TiO2-PEG NPs injection alone does not affect tumor development. However, upon NIR irradiation of the NPs injected tumor, edemas appear on the tumors within 3 d due to thermal damage. On the fifth day, the tumors shrink, and black scars are left on the tumor sites. On the 14th day, the tumors disappear, leaving only smooth scars on the original tumor sites. These results clarify that H-TiO2-PEG NPs are an effective PTT agent for in vivo cancer therapy. Change of body weight is an important parameter for the evaluation of toxicity or damage during treatment. Consequently, the body weights of mice were recorded during the therapy period. As shown in Figure 8b, in the first 3 d, body weights of mice decrease slightly in laser group, NPs group, and laser + NPs group, but increase slightly in control group. This is likely due to the fact that the mice were anesthetized during this period of time, which negatively affects food consumed and thus losses body weight slightly. However, mice in control group were not anesthetized so their body weights increase. From 4th to 14th day, body weights of all mice increase, which demonstrates that the PTT agent and treatment used in this study are nontoxic and safe to tumor-bearing mice. However,


body weight in laser + NPs group is significantly less than the other three groups after the 4th day, due to tumor shrinkage as measured by reduction in tumor volume. To further evaluate the PTT performance of H-TiO2-PEG, survivals of the mice after treatment were also recorded. As shown in Figure 8c, mice live healthily for more than 50 d in laser + NPs group. However, some mice in the other three groups die from days 25 to 34, and all of the mice in these groups die after day 47. These results demonstrate that H-TiO2PEG NPs, as high-performance PTT agents, are promising for further biomedical application.

3. Conclusion In summary, H-TiO2 NPs were explored as a new near infrared triggered photothermal agent for cancer therapy. We have demonstrated the photothermal effect of H-TiO2 NPs can be attributed to its dramatically enhanced nonradiative recombination, which lead to an excellent performance of photothermal conversion in the NIR range. Our results also demonstrate that



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H-TiO2 NPs coated by PEG possess several features: 1) high NIR photothermal effect, 2) low toxicity and good biocompatibility, and 3) low cost and facile synthetic method. More importantly, H-TiO2-PEG NPs are successfully used as photothermal agent for NIR-triggered cancer therapy both in vitro and in vivo. This work also provides an experimental basis for the promising application of H-TiO2-PEG NPs as an effective photothermal agent for cancer therapy. It is demonstrated that NIR-triggered cancer photothermal therapy of H-TiO2 shows better applicability than UV light–induced photodynamic therapy of TiO2 and thus can advance the applications of TiO2 in biomedicine in future.

4. Experimental Section Preparation of H-TiO2 Nanoparticles: Commercial TiO2 nanoparticles (Degussa P25) were purchased from Sigma-Aldrich. 0.10 g TiO2 NPs were dispersed in 50 mL ethanol, and then drop-casted onto a 6 inch-Si wafer. The drop-casting process was repeated several times to achieve a TiO2 mass loading of 0.5–0.6 mg cm−2 (100–150 mg on a whole wafer). This wafer was then transferred into a chamber for the hydrogenation treatment with H2 plasma, and there an instrument of inductively coupled plasma (ICP) (Plasmalab 100 ICP-CVD, Oxford Instruments) was used. The H2 plasma treatment was performed at 150 °C for 20 min. The ICP power was 3000 W, the chamber pressure was 25.8–27.1 mTorr, and the H2 flow rate was 50 sccm. After this treatment, hydrogenated TiO2 (H-TiO2) NPs was obtained and scratched from the Si wafer. IR Absorption and Photothermal Principle of H-TiO2 NPs: X-ray diffraction (XRD) pattern of the samples was recorded on a diffractometer (SIEMENS D5000) with Cu-K radiation. The optical absorption in the range from UV to the NIR was measured by a diffuse reflectance accessory of a UV–vis–NIR spectrometer (Cary 5000). EPR spectra were recorded at the temperature of 77 K using a Bruker BioSpin CW X-band (9.5 GHz) spectrometer (ELEXYS E500). PL spectroscopy was performed by using a Czerny–Turner spectrograph (Jobin Yvon SPEX 1000M) with a focal length of 1000 mm. The excitation wavelength of 266 nm was generated by a femtosecond laser (Coherent MIRA 900-F) followed by a pulse picker (Coherent Pulse Picker) and a third harmonic generator (APE HarmoniXX THG). PEG Coating and Photothermal Conversion Efficiency of H-TiO2-PEG NPs: For biomedical applications, the H-TiO2 should be disperse and stable in serum. In this study, PEG (molecular weight 1500) was used to enwrap the H-TiO2 NPs to improve their stability. 20 mg of H-TiO2 powder was dispersed in 75 mL ethanol by an ultrasound treatment of 30 min. The H-TiO2 contained ethanol was dropped into a 25 mL PEGethanol solution (20 mg mL−1 of PEG), and stirred for 24 h. H-TiO2-PEG NPs were separated by centrifugation, and were washed with ultrapure water. The as-prepared H-TiO2-PEG NPs were dispersed in the ultrapure water and stored at 4 °C. Micromorphologies of H-TiO2 before and after PEG coating were investigated at the same concentration through a transmission electron microscope (FEI Tecnai F20). Size distribution and zeta potential of H-TiO2 and H-TiO2-PEG were measured by a particle size zeta potential analyzer (Nano ZS, Malvern Instruments Ltd, England). The UV–visible spectra of H-TiO2 and H-TiO2-PEG were determined by using an UV–visible spectrophotometer (T10CS, Beijing Purkinje General Instrument, China). For evaluating the photothermal conversion efficiency of H-TiO2-PEG, 2 mL aqueous dispersion of H-TiO2-PEG (100 µg mL−1) were moved into a well of 24-well culture plate, and irradiated under an 808 nm NIR laser at a power density of 2 W cm−2 for 600 s. The temperature of the dispersion was measured every 60 s after the start of irradiation. After the laser irradiation was shut off, the temperature was further measured for another 1500 s with the same intervals. Ultrapure water as control group was treated under the same conditions. Photothermal conversion efficiency (η) of H-TiO2-PEG

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was then calculated according to the methods reported previously.[18] In order to investigate the stability, H-TiO2-PEG NPs were dispersed in sterile fetal bovine serum (FBS) solution at room temperature for 7 d, and size distributions of the nanoparticles were measured every day by the particle size zeta potential analyzer. Cell Culture and Cytotoxicity of H-TiO2-PEG NPs In Vitro: MCF-7 cell line of human breast cancer and 4T1 cell line of murine breast cancer were cultured in RPMI1640 medium and supplemented with 10% FBS. The cells were maintained at 37 °C incubator with 5% CO2. To evaluate the cytotoxicity of H-TiO2-PEG, MCF-7 or 4T1 cells were plated in 96-well plates (1 × 104 cells per well) and cultured for 24 h. The cells were incubated with different doses of H-TiO2-PEG (50–300 µg mL−1) for 24 h. The viability of cells was assayed by the MTT assay. Briefly, 10 µL of MTT (5 mg mL−1 in PBS) was added into every well, and incubated for 4 h. Next, DMSO was used to dissolve the formazan crystals. The absorbance was measured by a microplate absorbance reader (Biorad iMARKTM, USA), and the cell viability was calculated. Uptake of H-TiO2-PEG NPs by Cancer Cells: To investigate the uptake of H-TiO2-PEG in the cancer cells, fluorescent dye ARS was used to stain TiO2 as previously reported.[19] MCF-7 and 4T1 cells (2 × 105 cells) were seeded into 35 mm culture dishes and cultured for 24 h, respectively. The culture media was then replaced by fresh medium contained ARS (10 µg mL−1) or ARS-H-TiO2-PEG (100 µg mL−1). The cells were incubated for 2 h, then washed with PBS, and moved to tubes after incubation with trypsin-EDTA. After centrifugation and resuspension with PBS, red fluorescence of cells was analyzed by flow cytometer (FACSCalibur, BD, USA). Photothermal Therapy of H-TiO2-PEG NPs In Vitro: To quantitatively evaluate the photothermal therapy efficiency of H-TiO2-PEG on cancer cells, MCF-7 and 4T1 cells were seeded into 96-well plates (1 × 104 cells per well), respectively. The cells were then incubated with fresh DMEM and 100 µg mL−1 of H-TiO2-PEG containing DMEM for 2 h. After 2 h incubation, all of the media were replaced by fresh DMEM. The cells were then irradiated by an 808 nm NIR laser (2 W cm−2) for 0–5 min and were cultured for another 24 h. 10 µL of MTT was added into each well and incubated for 4 h. The MTT solution was removed and 100 µL DMSO was added to dissolve the formazan crystals. Finally, the absorbance was measured and the cell viability was calculated. In order to further evaluate the photothermal therapy of H-TiO2-PEG on cancer cells, MCF-7 or 4T1 cells were cultured in 35 mm dishes. The cells were then incubated with fresh DMEM containing 100 µg mL−1 of H-TiO2-PEG for 2 h. The culture media were then replaced by fresh DMEM, and the cells were irradiated by 808 nm NIR (2 W cm−2) for 5 min. The cells were stained with both calcein AM and PI. The live and dead cells were observed by confocal microscopy as previously described. Toxicity Evaluation of H-TiO2-PEG NPs In Vivo: In the animal experiments, the animal care and handing procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments at Ningbo University (Permit No. SYXK Zhe 2013-0191). For assessing toxicity of H-TiO2-PEG in vivo, 24 healthy Blab/C mice were divided into four groups randomly. The mice were intravenously injected with different doses (1, 5, 25 mg kg−1) of H-TiO2-PEG, respectively. Mice injected with saline were used as the control. Mice were observed for behavioral changes over one month period. After one month, all mice were sacrificed. Mice's blood were collected by a cardiac puncture method for hematological and were analyzed by blood analyzer (Sysmex XT-1800i, Japan) and Hitachi 7600-110 autoanalyzer (Hitachi, Tokyo, Japan). The main organs including heart, liver, spleen, kidney, and lung were preserved in a 10% formalin solution and stained with H&E for histological analysis to assess the toxicity of H-TiO2-PEG. Biodistribution of H-TiO2-PEG NPs In Vivo: For evaluating the distribution of H-TiO2-PEG in tumor-bearing mice, tumor model was established. 4T1 cells (1 × 106 cells for one mouse) suspended in 100 µL of serum free medium were inoculated subcutaneously in several female Balb/C mice (five weeks old). A digital caliper was used to measure the size of tumor. When the tumor grew to 3–4 mm, mice were intravenously injected with 100 µL of H-TiO2-PEG aqueous dispersion (2000 µg mL−1). The mice were sacrificed after 24 h, and the concentration of Ti in main




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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements W.R. and Y.Y. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (31170964, U1432114, U1332117, and 11475012), the Hundred Talents Program of Chinese Academy of Sciences (2010-735), the Natural Science Foundation of Zhejiang province (LY15C100002), and by the Natural Science Foundation of Ningbo city (2014A610166). Prof. Xiaoyuan Chen is appreciated for evaluating scientific sense of this study. The authors thank Prof. Qiang Yao for supplying of a photothermal imaging system. The authors also thank Mr. Fei Zhou in the Experimental Animal Center of Ningbo University for conducting animal feeding. Yong Yan is supported by means of a doctoral scholarship from the Carl Zeiss Stiftung (Germany). A minor change was made to the text in the second paragraph of Section 2.1 on July 15.

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organs were measured by ICP-MS (NexION 300, Perkin-Elmer, USA) as described previously.[18] Photothermal Imaging and Therapy of H-TiO2-PEG NPs In Vivo: Tumor models were established in 24 female Balb/C mice (five weeks old) as described above. A digital caliper was used to measure the size of tumor. Tumor volume = (tumor length) × (tumor width)2/2. When the tumors grew to 3–4 mm in diameter, the mice were randomly divided into four groups, and each group contained six mice. The mice were anesthetized by intraperitoneal injection of chloral hydrate solution (8 wt%), and were given an intratumor injection with 100 µL of H-TiO2-PEG aqueous dispersion (100 µg mL−1) or with 100 µL of saline. The tumor sites were irradiated with or without an 808 nm NIR laser at 2 W cm−2 for 5 min. The temperature change of tumor site was measured by a photothermal imaging system (Ti400, Fluke, USA) during NIR irradiation. Four mice were sacrificed after NIR irradiation; their tumors were collected for histological analysis to evaluate the nanoparticles triggered photothermal injury of tumor. In order to kill the residual tumor cells, the tumor sites were irradiated each of the following 2 d under the same conditions. The tumor sizes of mice were measured by a digital caliper for 14 d and the tumor volume was calculated according to the formula of tumor volume mentioned above. Relative tumor volumes were calculated as V/VO (VO was the tumor volume when the treatment was initiated). Body weight and survival of the mice were also recorded.

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