PDT

Accepted Manuscript Black Titania-Based Theranostic Nanoplatform for Single NIR Laser Induced DualModal Imaging-Guided PTT/PDT Dr. Juan Mou, Dr. Tianquan Lin, Prof. Fuqiang Huang, Prof. Hangrong Chen, Prof. Jianlin Shi PII:

S0142-9612(16)00011-9

DOI:

10.1016/j.biomaterials.2016.01.009

Reference:

JBMT 17285

To appear in:

Biomaterials

Received Date: 10 November 2015 Revised Date:

26 December 2015

Accepted Date: 1 January 2016

Please cite this article as: Mou J, Lin T, Huang F, Chen H, Shi J, Black Titania-Based Theranostic Nanoplatform for Single NIR Laser Induced Dual-Modal Imaging-Guided PTT/PDT, Biomaterials (2016), doi: 10.1016/j.biomaterials.2016.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Black Titania-Based Theranostic Nanoplatform for Single NIR Laser Induced Dual-Modal Imaging-Guided PTT/PDT

Dr. Juan Mou, Prof. Hangrong Chen, and Prof. Jianlin Shi

RI PT

Juan Mou1, Tianquan Lin2, Fuqiang Huang2, Hangrong Chen1
and Jianlin Shi1


State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

SC

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China.

M AN U

Dr. Tianquan Lin, Prof. Fuqiang Huang

CAS Key Laboratory of Materials for Energy Conversion,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China.

TE D


Corresponding authors: [email protected]; [email protected]

KEYWORDS: black titania, photoacoustic imaging, photothermal therapy, photodynamic

ABSTRACT

EP

therapy, single NIR laser

AC C

Substantially different from traditional combinatorial-treatment of photothermal therapy (PTT) and photodynamic therapy (PDT) by using multi-component nanocomposite under excitation of separate wavelength, a novel single near infrared (NIR) laser-induced multifunctional theranostic nanoplatform has been rationally and successfully constructed by a single component black titania (B-TiO2-x) for effective imaging-guided cancer therapy for the first time. This multifunctional PEGylated B-TiO2-x shows high dispersity/stability in aqueous solution, excellent hemo/histocompatibility and broad absorption ranging from NIR to ultraviolet (UV). Both in vitro and in vivo results well demonstrated that such a novel multifunctional theranostic nanoplaform could achieve high therapeutic efficacy of simultaneous and synergistic PTT/PDT under the guidance of infrared thermal/photoacoustic 1

ACCEPTED MANUSCRIPT (PA) dual-modal imaging, which was triggered by a single NIR laser. This research circumvents the conventional obstacles of using multi-component nanocomposites, UV light and high laser power density. Furthermore, negligible side effects to blood and main tissues could be found in 3 months’ investigation, facilitating its potential biomedical application. INTRODUCTION

RI PT

1.

In the past decades, phototherapy has aroused extensive interests as a powerful technique for cancer treatment, attributing to its convenience and minimal invasiveness [1-4].

SC

Photothermal therapy (PTT) [5-7] and photodynamic therapy (PDT) [8-10] are two main kinds of general phototherapy protocols, both of which involve the administration of

M AN U

photosensitizers with strong optical absorption property, to effectively convert light energy into hyperthermia or generate toxic reactive oxygen species (ROS) to kill cancer cells, respectively. The combination of these two modalities holds great promise to overcome respective limitations [11-14], such as oxygen dependence and less efficient therapeutic efficacy of PDT, as well as non-selectivity and requirement of high laser power density of

TE D

PTT, etc.. Currently, a number of nanomaterials, such as gold nanostructures [11, 15-18], carbon nanotubes [19], nano-graphene oxide [20, 21], and silica nanoparticles [22, 23], etc., loading various photosensitizers, such as Ce6, phthalocyanine, indocyanine green (ICG), and

EP

methylene blue, etc., have been used in the combinatorial treatment of PTT and PDT. However, such combined strategies generally suffer from inevitable systemic side effects

AC C

since two different functional components often require two seperated excitation wavelengths, consequently leading to prolonged and complicated treatment. Therefore, it is highly desirable to develop an efficient and single component nanomaterial rather than multi-component nanocomposites for simultaneous and synergistic PTT/PDT treatment. Recently, infrared thermal imaging [24-27] and photoacoustic (PA) imaging [28-30] have attracted much attention for cancer diagnosis. Infrared thermal imaging is a fast and sensitive imaging modality, while PA imaging is a newly developed noninvasive imaging modality combining high sensitivity and specificity of optical imaging as well as high spatial resolution of ultrasound imaging. PA imaging offers in vivo three-dimensional (3D) structural and 2

ACCEPTED MANUSCRIPT functional imaging of different biological tissues, thus has attracted extensive interests in preclinical research and clinical application [31, 32]. A variety of contrast agents with optical absorption have been used to amplify PA signal based on the thermal expansion [33-35]. Despite advances in developing PA imaging systems [31, 36, 37], the exploration of

RI PT

multifunctional contrast agents for enhancing PA amplitude at centimeter depths, and the combination with other imaging modalities for obtaining more accurate and comprehensive diagnosis information are still urgently needed. Therefore, the strategy of integrated infrared thermal/PA dual-modal imaging-guided simultaneous and synergistic PTT/PDT treatment

SC

will attract great interests in terms of substantially enhancing the accuracy, efficacy and safety of cancer treatment.

M AN U

The recently discovered black titania (B-TiO2-x), as a rising star, has attracted substantial interest for its potential application in the fields of photocatalysis, photovoltaics and fuel cells, etc., attributing to its efficient optical absorption property ranging from NIR to UV [38-42]. Very recently, a kind of hydrogenated black TiO2 NPs has been demonstrated to have high photothermal converstion efficiency and could be used in cancer photothermal therapy [43].

TE D

Nevertheless, single PTT strategy is liable to cause unexpected damages to normal tissues. Fortunately, titania also features the capability of converting laser energy into chemical energy for PDT associated with various ROS species generation, such as hydroxyl radical

EP

(OH•) [44], superoxide anion radical (O2•-) [45], and hydrogen peroxide (H2O2) [46], which can react with biological molecules and eventually cause severe cancer cell death [47-50].

AC C

However, the previously reported titania for PDT was triggered by UV light, which generally suffers from limited penetration depth and results in damages to healthy tissues. Therefore, how to construct the combinational PTT and PDT nanoplatform-triggered by a single NIR laser based on B-TiO2-x for the synergistic tumor treatment, to circumvent the obstacles of conventional UV light excitation and high laser power density utilization, is great desirable but challenging. Herein, we reported the successful construction of a novel single NIR laser-induced multifunctional theranostic nanoplatform, based on single component black titania (B-TiO2-x) for in vivo infrared thermal/PA dual-modal imaging-guided simultaneous PTT/PDT for the 3

ACCEPTED MANUSCRIPT first time. This multifunctional B-TiO2-x, which was rapidly synthesized via a mass producible low temperature aluminum reduction, followed by PEG5000-NH2 modification, presented excellent dispersity and stability both in water and physiological solution. Meanwhile, this BTiO2-x exhibits a strong optical absorption ranging from NIR to UV, which is believed

RI PT

attributable to the localized surface plasmon resonance (LSPR) [51, 52]. This feature provides opportunity of using a single NIR laser of 808 nm to trigger B-TiO2-x for simultaneously producing hyperthermia and toxic ROS for PTT and PDT at a relatively low laser power density (1 W cm-2), respectively. Moreover, infrared thermal and PA imaging can visualize

SC

the tumor site/dimension/morphology for pre-treatment guidance and real-time monitoring in the PTT/PDT treatment. Both in vitro and in vivo results well demonstrated that such

M AN U

multifunctional theranostic nanoplaform could achieve high efficient tumor treatment of synergistic PTT and PDT under the guidance of infrared thermal/PA dual-modal imaging, upon exposure to a single NIR laser irradiation. As far as we know, this is the first report of constructing the novel biocompatible B-TiO2-x to serve as a single NIR laser-induced multifunctional theranostic nanoplatform for infrared thermal/PA dual-modal imaging-guided

TE D

simultaneous PTT/PDT, which, as we believe, will open broad horizons for the biomedical applications of B-TiO2-x and exploration of imaging-guided therapy of cancers. RESULTS AND DISCUSSIONS

EP

2.

2.1 Synthesis and Characterization

AC C

The as-constructed novel single NIR laser-induced multifunctional theranostic nanoplatform, which is based on the single component B-TiO2-x for infrared thermal/PA dualmodal imaging-guided simultaneous PTT/PDT, is illustrated in Scheme 1.

4

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Scheme 1. Schematic illustration of as-constructed multifunctional single component B-TiO2-x for infrared thermal/PA dual-modal imaging-guided simultaneous PTT/PDT under a single NIR laser irradiation.

Pristine B-TiO2-x was synthesized via a mass producible low temperature aluminum

TE D

reduction process according to the previously reported [42]. The pristine B-TiO2-x suffers from poor dispersity in water and physiological solution, which can be apparently revealed by the instant precipitation of B-TiO2-x particles in physiological solution at the bottom of quartz

EP

cuvette when dispersion (the inset in Figure 1a). In order to obtain high dispersity and stability, PEG5000-NH2 is utilized to modify the surface to prevent particles aggregation under

AC C

sonication through the covalent bound of amine group with unsaturated Ti atom at the surface. The morphology and microstructure information of the pristine B-TiO2-x and PEGylated BTiO2-x (B-TiO2-x-PEG) were investigated by transmission electron microscopy (TEM) and high resolution-TEM (HR-TEM). As shown in Figure 1a and Figure S1a, the pristine B-TiO2-x displays a unique core-shell structure which is comprised of crystalline core of TiO2 with an average diameter of 25 nm (±4.5 nm) and an amorphous TiO2-x shell with an average thickness of 1.5 nm. The HRTEM image (Figure S1b) further confirms the amorphous shell and the well-resolved lattice fringes of ~0.35 nm, belonging to the (101) planes of anatase. The existence of Ti and O elements are obviously evidenced by energy dispersive 5

ACCEPTED MANUSCRIPT spectrometer (EDS) analysis (Figure S2). After PEGylation, no significant morphology changes were observed (Figure 1b). Moreover, all the diffraction peaks (Figure S3a) referenced to anatase (JCPDS 21-1272), could be found in both pristine B-TiO2-x and B-TiO2x-PEG,

indicating that no phase transformation occurs after surface modification. The Raman

RI PT

spectra shown in Figure S3b show no structural changes after PEGylation, since both of them exhibit the distinct Raman-active modes of anatase TiO2 phase with spectral overlap. It can be found that the hydrodynamic diameter of B-TiO2-x increases from 59 nm to 124 nm after PEGylation (Figure S4a, polydispersity index=0.192), and the corresponding Zeta potential

SC

moves towards positive direction (Figure S4b), indicating the successful surface modification. In addition, as shown in the Fourier transform infrared (FTIR) spectra (Figure S5), the

M AN U

appearance of Ti–N–O vibrating band at 1384 cm-1 in B-TiO2-x-PEG could be clearly verified, confirming the successful surface modification and enabling the potential bio-application of B-TiO2-x [53]. B-TiO2-x-PEG could be well dispersed in physiological solution and kept stable for at least one week without detectable aggregation, which can be evidenced by the unchanged hydrodynamic diameter and Zeta potentials of aqueous solution containing B-

TE D

TiO2-x-PEG placed for 3 and 7 days (Figure S4a-b), and the corresponding photograph for one

AC C

EP

week (the inset in Figure S4a).

6

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 1. Representative TEM images of the pristine B-TiO2-x (a) and B-TiO2-x-PEG (b), the insets show their corresponding digital photographs in physiological solution. (c) UV-vis absorbance spectra of aqueous solutions containing B-TiO2-x at varied concentrations and (d) the corresponding linear plot of absorbance vs concentrations at 808 nm. Infrared thermal images of water droplet and aqueous droplets containing BTiO2-x (concentrations of C1-0.16 and C2-0.64 mg mL-1, respectively) under laser irradiation for 60 s at varied power densities of 0.48 (e) and 0.78 (f) W cm-2, respectively. (g) Plots of the PA amplitude vs concentrations of aqueous solution containing B-TiO2-x under NIR laser irradiation. (h) Temperature evolution curves of pure water and aqueous solution containing B-TiO2-x at different concentrations under laser irradiation. (i) Photothermal stability investigation after laser irradiation for 10 cycles. (j) ESR spectra of different samples.

7

ACCEPTED MANUSCRIPT Apparently, B-TiO2-x (the modified B-TiO2-x-PEG is abbreviated hereafter into B-TiO2-x) shows a broad optical absorption property ranging from NIR to UV (Figure 1c) owing to the LSPR, since B-TiO2-x is a kind of p-type semiconductor with relatively high carrier (holes) concentrations, thus showing strong free carrier absorption. The absorption intensity at a

RI PT

preferable wavelength of 808 nm, which is a transparent window for tissues [54], linearly increases with elevated concentrations of B-TiO2-x (Figure 1d). The strong NIR absorption of B-TiO2-x promises an excellent infrared thermal/PA dual-modal imaging performance. Initially, the infrared thermal images of the water droplet and aqueous droplets containing B-

SC

TiO2-x at different concentrations were recorded at varied power densities and durations of NIR laser irradiation (Figure 1e-f). Water droplet shows a mild temperature increase and a

M AN U

negligible color change in the infrared thermal images. Whereas, the aqueous droplets containing B-TiO2-x show sharp temperature elevation and apparent color changes, presenting strong dependence on concentrations and power densities, suggesting that B-TiO2-x is capable of serving as an excellent contrast agent for infrared thermal imaging. More interesting, the PA signal intensity produced by B-TiO2-x is found to exhibit a significant linear enhancement

TE D

with concentrations (Figure 1g) via an agar-phantom study, with the linear correlation of R2 = 0.998. The gradually brightened PA images (the inset in Figure 1g) apparently indicate that BTiO2-x can be used as an excellent PA imaging contrast agent.

EP

Photothermal effect of B-TiO2-x was further examined by monitoring the temperature changes of aqueous solutions containing B-TiO2-x as functions of concentrations, laser power

AC C

density and irradiation time at the same preferable laser wavelength of 808 nm. Apparently, the temperatures of B-TiO2-x solutions increase from room temperature to 32.7-35.5 °C upon laser irradiation, exhibiting proportional dependence on concentrations (Figure 1h) and laser power density (Figure S6). While water as a control shows a slight increase to 28.7 °C. Notably, there is no photobleaching effect detected in B-TiO2-x solution after laser irradiation repeated for 10 cycles (Figure 1i), indicating an excellent photothermal stability of B-TiO2-x. Furthermore, in order to investigate the potential PDT activity of B-TiO2-x, a kind of photochemical method with 1,3-diphenylisobenzofuran (DPBF) as singlet oxygen (1O2) probe in acetonitrile was conducted to detect the generation of 1O2 [55-58]. In the presence of B8

ACCEPTED MANUSCRIPT TiO2-x, a noticeable reduction of absorption intensity of DPBF as a function of NIR laser irradiation time is observed, while the control group treated with NIR laser alone shows a slight decrease (Figure S7), indicative of the capability of B-TiO2-x to generate 1O2 under NIR laser irradiation. Additionally, the electron spin resonance (ESR) spectroscopy is also used to

RI PT

directly detect the generation of hydroxyl radicals (•OH) by using 5,5'-dimethylpyrroline-1oxide (DMPO) as spin trap agent. The appearance of 1:2:2:1 multiplicity in the ESR spectrum of B-TiO2-x evidences the characteristics of DMPO-OH adduct (Figure 1j), suggesting the generation of hydroxyl radicals, which is in good agreement with literatures [59, 60]. As a

SC

comparison, no production of •OH could be detected without B-TiO2-x. The production of •OH in B-TiO2-x without NIR laser irradition was mainly due to the effect of visible light from the

M AN U

lamp. Interestingly, the signals intensity sharply increases upon NIR laser irradiation, confirming that B-TiO2-x can efficiently promote the conversion of the NIR laser energy to chemical energy and generate ROS for PDT. Therefore, as we expected, this single component B-TiO2-x can serve as a new multifunctional theranostic nanoplatform for the following infrared thermal/PA dual-modal imaging-guided simultaneous PTT/PDT under a

TE D

single NIR laser irradiation.

2.2 Cytotoxicity, intracellular distribution and Phototherapy of B-TiO2-x in vitro Prior to its bio-application in vivo, the cell viability was evaluated by a standard 3-(4,5-

EP

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Since HeLa cell line is an immortal and widely used cell line in scientific research, and suitable for cervical cancer

AC C

model establishment [61], HeLa cells were prefered for the following in intro and in vivo study. Firstly, HeLa cells were incubated with B-TiO2-x at varied concentrations for 24 h and 48 h, respectively. This B-TiO2-x shows negligible effect on the survival of HeLa cells, even at a high concentration up to 1 mg mL-1 with prolonged incubation time of 48 h (Figure S8, the black and red histograms). The low cytotoxicity was further evidenced by the unchanged cell morphology (Figure S9). In addition, as nanoparticles trend to be accumulated to liver, kidney and potential toxic to brain, three types of normal cells including rat liver cells (BRL), renal tubular duct epithelial cells of rat (NRK-52E) and brain capillary endothelial cells (BCECs) were selected for the cytotoxicity evaluation. After 24 incubation, the percentages of cell 9

ACCEPTED MANUSCRIPT viabilities of all the three types were above 85% (Figure S10a) and there were no significant changes in cells morphologies (Figure S10c-e), indicating B-TiO2-x is biocompatible to liver, kidney and brain. Furthermore, in order to make it more convinced and focus on the cytotoxicity of B-TiO2-x on brain, standard MTT assays were performed on two types of

RI PT

brain-related cancer cells, including human glioblastoma cells (U87MG) and rat adrenal pheochromocytoma cell line (PC12), which were generally used to get more information about diseases of brain. Similarly, after incubation with B-TiO2-x at varied concentrations for 24 h, no cytotoxicity against these two types of cancer cells could be found, evidenced by the

SC

cell viabilities all above 85% (Figure S10b) and unchanged cell morphologies (Figure S10f-g). Subsequently, the cellular uptake of B-TiO2-x at varied durations by confocal laser scanning

M AN U

microscopy (CLSM) was further investigated. It is clear that most of RITC (rhodamine B isothiocyanate)-labeled B-TiO2-x are in the cytoplasm of HeLa cells after both 1 h (Figure S10a) and 4 h (Figure S10b) co-incubation, and the brighter red fluorescence originating from RITC and representing B-TiO2-x after 4 h incubation evidenced the enhanced cell uptake with prolonging incubation time.

TE D

However, when HeLa cells were incubated with B-TiO2-x at varied concentrations for 4 h and then exposed to a NIR laser irradiation (808 nm, 1Wcm-2, 5 min), the viability and proliferation of HeLa cells were substantially inhibited (Figure S8, the blue histogram).

EP

Trypan blue staining was also performed to visually demonstrate the cell death. As shown in Figure 2a1-a3, the control groups including HeLa cells treated with culture medium, NIR

AC C

irradiation alone, and B-TiO2-x incubation alone display negligible changes, whereas HeLa cells treated with B-TiO2-x+NIR laser at a power density of 1 W cm-2, rather than previously reported 2 W cm-2 for 5min [43], are stained in blue (Figure 2a4) and presented severe cell death, which is attributable to the simultaneous and synergistic generation of hyperthermia and toxic ROS in presence of B-TiO2-x and NIR laser irradiation. The phototherapy effect was further investigated by CLSM. Under 488 nm laser irradiation, the viable cells in green by calcein-acetoxymethyl (calcein-AM) labeling could be detected in the above-mentioned control groups (Figure 2b1-b3). In contrast, dead cells in red by propidium iodide (PI) staining could be found in the group treated with B-TiO2-x+NIR laser irradiation (Figure 2b4). 10

ACCEPTED MANUSCRIPT Additionally, to further verify the PDT effect in inducing cell death, 2,7-dichloro-dihydrofluorescien diacetate (DCFH-DA), which can be rapidly oxidized into highly green fluorescent 2,7-dichlorofluorescein (DCF) in the presence of ROS, was utilized to detect the production of ROS. Negligible green fluorescence can be observed in the above mentioned

RI PT

control cell groups, indicating negligible generation of ROS, as shown in Figure 2c1-c3. However, significantly strong green fluorescence originated from DCF can be detected in the cell group of B-TiO2-x+NIR laser irradiation (Figure 2c4), definitely proving the efficient generation of ROS and PDT activity of B-TiO2-x. The bright and overlay images demonstrate

SC

the existence of HeLa cells (Figure 2d-e). Furthermore, the green fluorescent intensities were quantified, as shown in Figure S12. It is well demonstrated that the green fluorescent intensity

M AN U

in the cell group treated with B-TiO2-x+NIR laser irradiation is much stronger (about 5-6 folds) than that in the aforementioned control groups, indicating that B-TiO2-x can effectively produce ROS under the NIR laser irradiation. These in vitro results confirm that this single component B-TiO2-x possesses the capability of simultaneously converting NIR laser energy into both hyperthermia and chemical energy to synergistically kill cancer cells at a relatively

TE D

low laser power density, which is much beneficial for the improved therapeutic safety and

AC C

EP

decreased dose of materials.

11

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 2. Optical microscopic images (a) of HeLa cells stained by trypan blue, confocal fluorescence

EP

images of HeLa cells co-stained by calcein-AM and PI (b), DCF (c), bright (d) and overlay (e) channels after different treatments: (a1-e1): without treatment as a control; (a2-e2): NIR laser irradiation alone for 5

AC C

min (808 nm, 1 W cm-2); (a3-e3): B-TiO2-x incubation alone (0.25 mg mL-1); (a4-e4): B-TiO2-x+NIR laser irradiation. All the scale bars in (a-e) are 50 µm.

2.3 Investigation on the Long-Term Toxicity in vivo Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in many applications. Recently, their biomedical applications inculding drug delivery, cell imaging, photodynamic and sonodynamic therapy for cancer treatment, biosensors for biological assay, and genetic engineering, etc., have attracted extensive interests [62]. Meanwhile, many studies have been carried out to investigate the potential toxicological mechanism of TiO2 NPs [63]. It is well known that the toxicity of nanomaterials depends on their particle size, morphology, surface 12

ACCEPTED MANUSCRIPT charge, structure, composition, injection dose, biological surroundings, etc. Due to the versatility of TiO2 NPs in terms of above mentioned factors, it’s hard to definitely draw a conclusion regarding the toxicity of TiO2 NPs [64, 65]. Therefore, a detailed in vivo investigation of the biomedical potential of B-TiO2-x was conducted as follows. The mice after

RI PT

intravenous injection of B-TiO2-x were euthanized at predetermined time points (i.e., 3, 30, 90 days). The blood was acquired for hemanalysis, and main organs (including heart, liver, spleen, lung and kidney) were stained with hematoxylin and eosin (H&E) for histological analysis, respectively. No disorder is detected in a series of blood indexes, including key

SC

biochemistry parameters, liver and kidney function indexes (Figure 3a-o). And the tissues show no abnormity from the H&E staining results after injection of B-TiO2-x (Figure 3p),

M AN U

indicaiting that B-TiO2-x has no significant damages to blood and main tissues. Moreover, in order to investigate the potential in vivo brain toxicity, the brain tissue sections of cortex, hippocampus and striatum, which were collected from the mice treated with intravenous injection of B-TiO2-x at a same Ti dose of 10 mg kg-1 for 3, 10 and 20 days, respectively, were stained with H&E for toxicity assessment. Compared with the control group, there are no

TE D

obvious tissue damages or any other side effects to aforementioned brain tissues (Figure S13), indicating that B-TiO2-x can be regarded as safe and non-toxic to brain. In brief, all the results have demonstrated that B-TiO2-x is highly hemo/histocompatible for further biomedical

AC C

EP

imaging and cancer treatment.

13

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 3. The variations of blood indexes (a-o) and time courses of histological changes in main organs of Kunming mice via H&E staining (p) after intravenous injection of physiological saline (control) or B-TiO2-x (10 mg Ti/kg) at different time points (i.e., 0, 3, 30, 90 days). All the scale bars in (p) are 50 µm.

2.4 In vivo Blood Circulation and Biodistribution Before in vivo imaging-guided therapy, the in vivo pharmacokinetic behavior of B-TiO2-x was investigated by inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis. As shown in Figure 4a, the blood circulation half-time of B-TiO2-x was calculated to be 1.03 h. After 24 h intravenous injection, large amount of B-TiO2-x appeared in liver and spleen, which was due to the reticuloendothelial system (RES) clearance. Around 2% of B14

ACCEPTED MANUSCRIPT TiO2-x was accumulated into tumor through the enhanced permeability and retention (EPR) effect (Figure 4b). It is noted that the accumulation efficiency could be further improved after targeting-ligands modification. B-TiO2-x could be passively targeted to tumor and determine the tumor site/dimension/morphology by infrared thermal/PA dual-modal imaging to guide

M AN U

SC

RI PT

the simultaneous PTT/PDT.

Figure 4. (a) Pharmacokinetic profile of B-TiO2-x following intravenous administration. (b) Biodistribution of B-TiO2-x at 24 h after intravenous injection in mice. Data represent means ± standard deviations.

2.5 Imaging-Guided Therapy in vivo

Motivated by the excellent infrared thermal/PA dual-modal imaging response and

TE D

PTT/PDT performance of B-TiO2-x in vitro, its feasibility for in vivo imaging-guided tumor therapy was then investigated. Firstly, mice bearing two tumors on each flank region were randomly divided into two groups (n=6) for different treatments: the first group and the

EP

second group were treated with intravenous injection of saline and B-TiO2-x, respectively, and NIR laser irradiation was applied on the left flank tumor. Taken the cytotoxicy, tumor

AC C

accumulation efficiency and theranostic efficacy into consideration, the injection dose of BTiO2-x was determined to be 10 mg kg-1 (0.25 mg mL-1, 80 µL). Infrared thermal images were recorded during the NIR laser irradiation. As expected, inconspicuous contrast enhancement can be found in the first group without B-TiO2-x administration (Figure 5a1-a4). However, significant contrast enhancement were achieved in the second group in the presence of BTiO2-x+NIR laser irradiation (Figure 5b1-b4), demonstrating that B-TiO2-x played an important role in enhancing infrared thermal imaging. Furthermore, three-dimensional (3D) ultrasound (US) and PA images of tumor at different time intervals (i.e., pre, 5 s, 1, 2, 3, and 4 h post intravenous

injection

of

B-TiO2-x)

were 15

acquired

for

determining

the

tumor

ACCEPTED MANUSCRIPT site/dimension/morphology (Figure 5c-e). Structural information of mouse anatomy can be discriminated in the US images (Figure 5c1-c6). Much higher resolution of contrast capability and distribution of B-TiO2-x within tumor can be further revealed by PA imaging. The PA signal shows a sharp increase in the first 5 s post-injection of B-TiO2-x with the strongest PA

RI PT

signal intensity gradually decreases afterwards (Figure 5d-e, Figure S14), mainly attributing to the clearance of B-TiO2-x from bloodstream during the circulation. In 4 h post-injection, the PA signal intensity retains averagely 3.5-folds stronger than that of pre-injection, indicating the as-prepared B-TiO2-x can be used as a good contrast agent for PA imaging for therapy

AC C

EP

TE D

M AN U

SC

guidance.

Figure 5. Infrared thermal images of nude mice bearing HeLa tumors after different treatment at varied time intervals: (a1-a4) NIR laser irradiation alone; (b1-b4) B-TiO2-x+NIR laser irradiation for 5 min (808 nm, 1 W cm-2). Three-dimensional US (c) and PA images (d) along with their overlay images (e) of the tumor sites acquired before and after intravenous injection of B-TiO2-x at different time intervals (i.e., pre, 5 s, 1, 2, 3, and 4 h). All the scale bars in (c-e) are 5 mm.

Inspired by the excellent in vitro performance of phototherapy effect, we further studied the simultaneous and synergistic PTT/PDT therapeutic efficacy of B-TiO2-x in vivo under the guidance of PA imaging and real-time monitoring by infrared thermal imaging. Under NIR irradiation at a relatively low power density of 1 W cm-2, negligible temperature elevation is 16

ACCEPTED MANUSCRIPT detected in the mice group treated without B-TiO2-x (Figure 5a1-a4). Comparatively, a significant temperature increase can be found in the mice group treated with B-TiO2-x+NIR laser irradiation (Figure 5b1-b4), which is high enough to induce severe cell death. Moreover, it is noteworthy that the elevated temperature has a positive impact on the photodynamic

RI PT

efficacy, since it could improve the partial oxygen pressure [66] and increased the reactivity of ROS [67]. Therefore, the combination of PTT and PDT are indeed expected to induce the synergistic and enhanced therapeutic effect. Upon NIR irradiation, the region of tumor treated with B-TiO2-x+NIR laser irradiation became whitish (Figure S15a-b) and then turned into

SC

black scars (Figure S15c) in 1 day, suggesting the disruption of blood perfusion. The damage to tumor tissue was further evidenced by H&E staining. The cells in tumor tissues treated with

M AN U

saline, B-TiO2-x or NIR alone remained unchanged in morphology (Figure 6a1-a3). However, cell shrinkages, nucleus loss and considerable karyolysis (Figure 6a4), which are the characteristics of dead cells, can be found in the tumor tissue treated with B-TiO2-x+NIR laser irradiation, indicating the efficient simultaneous and synergistic PTT/PDT therapeutic effect. Furthermore, the tumor growth rates of different groups were measured to evaluate the single

TE D

NIR laser-induced simultaneous PTT/PDT therapeutic efficacy. The tumors of mice group treated with B-TiO2-x+NIR laser irradiation is completely eliminated in 2 days (Figure 6b), whereas all other control groups display continuous tumor growth. The photographs (Figure

EP

6c1-c2) of mice bearing tumors by different treatments in 20 days visually confirm the reliable and high synergistic PTT/PDT therapeutic effect of B-TiO2-x in vivo. It is expected that, this

AC C

B-TiO2-x based multifunctional theranostic nanoplaform, triggered by a single NIR laser at a low NIR power density, can significantly impove the therapeutic efficiency and safety.

17

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 6. (a) H&E staining of tumor sections collected from the groups of mice upon different post

M AN U

treatments: (a1) saline; (a2) NIR laser irradiation alone; (a3) B-TiO2-x alone; and (a4) B-TiO2-x+NIR laser irradiation. (b) Tumor growth curves of different groups of tumors-bearing mice. (c) Photographs of mice in 20 days post-treatment. All the scale bars in (a) are 50 µm.

3 CONCLUSION

In summary, a novel single NIR laser-induced multifunctional theranostic nanoplatform

TE D

based on single component B-TiO2-x has been successfully constructed for dual-modal imaging-guided cancer therapy both in vitro and in vivo for the first time. This PEGylated BTiO2-x has several distinctive advantages, including: i) excellent dispersity and high stability in

EP

water and physiological solution; ii) enhanced infrared thermal/PA dual-modal imaging for pre-treatment guidance and real-time monitoring; iii) simultaneous and synergistic PTT/PDT

AC C

treatment triggered by a single NIR laser (808 nm) at a relatively low power density of 1 W cm-2; iv) low potential toxicity resulting from single component rather than complicated nanocomposites. In the presence of B-TiO2-x, significant enhanced contrast of infrared thermal imaging and amplified PA signal intensity can be achieved. More importantly, the tumor can be completely eliminated in 2 days under a low NIR laser power density of 1 W cm-2, and no recurrence can be found in 20 days’ investigation, attributing to the simultaneous and synergistic PTT/PDT effect. This work broadens the applicaion of B-TiO2-x from traditional energy- and environment-related fields to biomedical area and highlights its excellent performance, which can be developed as a new multifunctional theranostic nanoplatform for 18

ACCEPTED MANUSCRIPT dual-modal imaging-guided simultaneous PTT/PDT. Meanwhile, the NIR laser energyconversion efficiency and the long-term toxicity of B-TiO2-x remain to be meticulously investigated in the near future. 4

EXPERIMENTAL SECTION

RI PT

4.1 Preparation of pristine B-TiO2-x

Pristine B-TiO2-x was rapidly mass-produced via a low temperature aluminum reduction according to previously reported. Generally, TiO2-x sample and aluminum were placed in a

SC

two zone tube furnace, separately, and then evacuated to a base pressure lower than 0.5 Pa. After that, aluminum was heated at 800 °C, and TiO2-x sample was heated at 600 °C for 6 h.

4.2 Surface modification of B-TiO2-x

M AN U

Then, the Al-TiO2-x sample was post-annealed at 800 °C in an argon atmosphere for 12 h.

The as-prepared pristine B-TiO2-x suffered from poor water dispersity, which limited their biological application. PEG5000-NH2 was applied to modify the surface of B-TiO2-x under sonication. In brief, to the aqueous solution containing pristine B-TiO2-x, PEG5000-NH2 was

TE D

added. Then the mixture was sonicated by an ultrasonic cell disruptor in an ice bath. Finally, the product was centrifuged and washed several times with pure water, followed by redispersing in water for later use.

EP

4.3 Photothermal effect of B-TiO2-x in vitro

To explore the photothermal effect induced by B-TiO2-x under NIR irradiation, pure water

AC C

and aqueous solutions (2 mL) containing B-TiO2-x at varied concentrations (i.e., 0.16, 0.32, 0.64 mg mL-1) were put in quartz cuvettes and exposed to NIR laser irradiation for 5 min at different power densities (i.e., 0.35, 0.71, 1.05, 1.41, 1.77, 2.12, 2.47 W cm-2). The temperatures of the solution were recorded per 1 s by a thermocouple microprobe, which was submerged in the solution and perpendicular to the optical path to avoid NIR laser irradiation. For the photothermal stability study, the aqueous solution containing B-TiO2-x was subjected to NIR laser irradiation (808 nm, 1 W cm-2, 5 min) and repeated for 10 cycles.

19

ACCEPTED MANUSCRIPT 4.4 PDT activity of B-TiO2-x in vitro B-TiO2-x was dispersed in acetonitrile with 1, 3-diphenylisobenzofuran (DPBF), which was used as a chemical probe for singlet oxygen sensitization. The mixture was irradiated by NIR laser of 808 nm for 10 min and repeated for 6 times. The decreased absorbance of DPBF

RI PT

at 410 nm was monitored by UV–Vis spectrometer. For comparison, the ROS production of acetonitrile solution without B-TiO2-x was also measured in parallel under the same condition. 4.5 ESR measurements

The ESR spectra were recorded at 25 °C on a Bruker ESP-300E spectrometer. Prior to

SC

measurement, the solution containing B-TiO2-x was mixed with DMPO (100 mM, 40 µL), which was a spin trap agent for hydroxyl radicals (•OH), followed by being injected into

M AN U

quartz capillaries and illuminated with 808 nm laser for ESR measurement. 4.6 Cell culture and cytotoxicity of B-TiO2-x

HeLa cells, renal tubular duct epithelial cells of rat (NRK-52E), human glioblastoma cells (U87MG) and rat adrenal pheochromocytoma cell line (PC12) were suspended to a standard culture medium containing Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine

TE D

serum, and 1% antibiotic solution (GIBCO, Invitrogen) with a concentration of 105 cells mL-1 and cultured at 37 °C with atmosphere of humidified 5% CO2. Rat liver cells (BRL) and brain capillary endothelial cells (BCECs) were culutured in Roswell Park Memorial Insitute

EP

medium (RPMI) 1640 supplemented with 10% fetal bovine serum, and 1% antibiotic solution

AC C

at 37 °C with atmosphere of humidified 5% CO2. The cells were visually examined by optical microscopy and cultured to adhere for 24 h. Cell viability was evaluated by standard 3-(4,5)dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. The cells were incubated in a 96-well plate with culture medium in the presence of B-TiO2-x at different concentrations (i.e., 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 mg mL-1). After incubation for 24 h and 48 h, respectively, the culture medium was replaced with culture medium containing MTT (0.5 mg mL-1) and incubated for another 4 h. DMSO (100 µL) was added to dissolve the MTT-formazan formed by metabolically viable cells and shaken for homogeneity. The

20

ACCEPTED MANUSCRIPT absorbance was then measured on an enzyme-linked immuno sorbent assay (ELISA) reader at a test wavelength of 490 nm. Each test was repeated at least three times. 4.7 Simultaneous PTT/PDT therapeutic efficacy in vitro by MTT For investigate the simultaneous PTT/PDT ablation of cancer cell by MTT, HeLa cells

RI PT

were pre-cultured in 96-well cell culture plates (1×104 cells per well) for 24 h to adhere and then incubated with B-TiO2-x at a series of concentrations. After incubation for 4 h, cells were irradiated by 808 nm laser at a power density of 1 W cm-2 for 5 min and standard MTT assay was performed to determine the cell viabilities compared with the untreated control cells.

SC

4.8 Trypan blue staining

HeLa cells were incubated at 37 °C in a humidified 5% CO2 atmosphere until reaching

M AN U

70–80% confluence in cell culture plates, followed by adding the culture medium (1 mL) containing B-TiO2-x (0.25 mg mL-1) and co-incubated for 4 h. The cells were then irradiated with 808 nm laser. After NIR irradiation, HeLa cells were stained with trypan blue (4%) for optical imaging, while cells stained in blue were account for dead cells. 4.9 Investigation of simultaneous PTT/PDT therapeutic efficacy in vitro by CLSM

TE D

HeLa cells were seeded in confocal laser scanning microscope (CLSM) culture dishes and incubated in culture medium at 37 °C in a humidified 5% CO2 atmosphere. After 12 h incubation for reaching 80%-90% confluence, culture medium of B-TiO2-x (0.25 mg mL-1)

EP

was added and then incubated for another 4 h for cell uptake. Prior to laser irradiation, culture medium containing B-TiO2-x was replaced by fresh culture medium. After laser exposure, the

AC C

culture medium was removed and washed with PBS (phosphate buffer solution) for several times. Then the cells were co-stained with calcein-AM and PI solution for 15 min to distinguish live and dead cells. Live cells were stained in green caused by calcein-AM and dead cells in red caused by PI, respectively. 4.10

PDT effect of B-TiO2-x in vitro by CLSM

HeLa cells were seeded into the cell culture plates and incubated with B-TiO2-x for 12 h and then the cell culture medium was replaced with 2, 7-dichloro-dihydro-fluorescien diacetate (DCFH-DA) solution and incubated for another 30 min. The cell culture medium was removed and cells were then irradiated with 808 nm laser for 5 min. Cells were then 21

ACCEPTED MANUSCRIPT observed by monitoring the green fluorescence, which was attributing to the transformation of DCFH-DA into highly fluorescent 2,7-dichlorofluorescein (DCF) in the presence of reactive oxygen species (ROS), using the FITC channel in CLSM (FV1000, Olympus, Japan). 4.11

Infrared thermal imaging in vitro and in vivo

RI PT

In order to verify the contrast capability of B-TiO2-x for infrared thermal imaging in vitro, water droplet and aqueous droplets containing B-TiO2-x (C1: 0.16 mg mL-1, C2: 0.64 mg mL1

) were placed on the same plate and irradiated with an 808 nm laser for 60 s. The temperature

evolution and infrared thermal images were recorded on a digital infrared thermal image

SC

instrument (FLIRA325 sc, USA). For in vivo investigation, prior to infrared thermal imaging, mice bearing HeLa tumors were firstly anaesthetized and held at normal body temperature.

M AN U

Physiological solution containing B-TiO2-x was intravenously administrated to mice. The spatial temperature distributions of tumors and infrared thermal images were recorded during NIR laser irradiation (808 nm, 1 W cm-2, 5 min). 4.12

Photoacoustic imaging in vitro and in vivo

To investigate the PA imaging contrast capability of B-TiO2-x, agarose solutions in the

TE D

presence of B-TiO2-x at different concentrations were cool down at room temperature to solidify and create cylinders with an average diameter of 7 mm into phantoms. A complete PA image of agar-phantom was obtained at the wavelength of 808 nm in 5 nm steps. To

EP

analyze the images, a 2D cylindrical region of interest (ROI) at the full size of agar-phantom was drawn over the agar-phantom containing B-TiO2-x and the mean signal in the agar-

AC C

phantom was quantified. Finally, the linear plot of the PA signal as a function of B-TiO2-x concentrations was acquired. Female nude mouse bearing HeLa tumors was used for the in vivo PA imaging studies. The mouse scanned in the PA imaging system was fully anesthetized using isoflurane delivered through a nose-cone during the experiment. Prior to PA scan, the ROI of tumor was covered with a thin layer of coupling agent to stabilize the region and minimize any breathing and other motion artifacts. A saran-wrap water tank full of water was placed on top of the coupling agent. An ultrasonic transducer was placed in the water bath, which was therefore acoustically coupled to the ROI of tumor. The ultrasonic transducer can move freely in 3D 22

ACCEPTED MANUSCRIPT without applying any physical pressure on the mouse in this setup. PA images with lateral step size of 0.5 mm was acquired using a 5 MHz transducer at the wavelength of 808 nm. Along with the PA scan, ultrasound images were acquired using a 25 MHz transducer and the two images were then merged. PA and ultrasound images were acquired before and after

RI PT

injection of B-TiO2-x at different time intervals (i.e., pre, 5 s, 1, 2, 3 and 4 h post injection). Quantification of PA signals was done by drawing a 3D ROI over the tumor volume. 4.13

Simultaneous PTT/PDT therapeutic efficacy in vivo

For evaluating the simultaneous PTT/PDT therapeutic efficacy in vivo, the mice bearing

SC

HeLa tumors on two flanks were randomly divided into two groups (n=6): the first and the second groups were treated with intravenous injection of saline and B-TiO2-x at a dose of 10

M AN U

mg kg-1 (0.25 mg mL-1, 80 µL), respectively, and laser irradiation was applied on the left flank tumor. Prior to irradiation, the mice were anesthetized and the temperature of tumor center was real-time monitored by an infrared thermal imaging instrument (FLIR sc325). The tumor size was recorded by a digital caliper every 5 days. Animal experiments

TE D

4.14

All animals including female nude mice and Kunming mice were purchased and raised at animal laboratory of Tongji University. Nude mice bearing HeLa tumors on two flanks region were established by subcutaneously injection of HeLa cells on two flanks region, which were

EP

used for blood circulation, biodistribution, infrared thermal imaging, PA imaging, and

AC C

simultaneous PTT/PDT study. Kunming mice were used for hemo/histocompatibility investigation. B-TiO2-x was intravenously administrated into mice. All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by Tongji University Animal Studies Committee. 4.15

Statistics

Results of this study are displayed as the mean ± standard deviation of at least three independent measurements. Unpaired two-tailed Student’s t-test was applied for all statistical evaluations. P-value less than 0.05 was considered to be statistically significant.

23

ACCEPTED MANUSCRIPT AUTHOR CONTRIBUTIONS The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

RI PT

SUPPLEMENTARY MATERIALS Supplementary materials may include Figures S1-S15. ACKNOWLEDGEMENTS

SC

This work was supported by the National Basic Research Program of China (973 Program, Grant No.2011CB707905), China National Funds for Distinguished Young Scientists

M AN U

(51225202), National Natural Science Foundation of China (Grant No. 51132009), Shanghai

AC C

EP

TE D

Excellent Academic Leaders Program (Grant No.14XD1403800).

24

ACCEPTED MANUSCRIPT Table of contents A novel single near infrared (NIR) laser-induced multifunctional theranostic nanoplatform based on single component black titania (B-TiO2-x) has been successfully constructed for in vivo effective dual–modal imaging-guided cancer therapy for the first time.

RI PT

This highly biocompatible B-TiO2-x with PEGylation shows broad optical absorption property ranging from NIR to ultraviolet (UV), and has been demonstrated both in vitro and in vivo, to be capable of serving as a new multifunctional theranostic nanoplatform for infrared thermal/photoacousitc (PA) dual-modal imaging-guided simultaneous photothermal therapy

SC

(PTT) and photodynamic therapy (PDT), upon a single NIR laser irradiation.

therapy, single NIR laser

M AN U

Keywords: black titania, photoacoustic imaging, photothermal therapy, photodynamic

Black Titania-Based Theranostic Nanoplatform for Single NIR Laser Induced Dual-Modal Imaging-Guided PTT/PDT

AC C

EP

TE D

Juan Mou, Tianquan Lin, Fuqiang Huang, Hangrong Chen
and Jianlin Shi


25

ACCEPTED MANUSCRIPT References [1] Bown SG. Phototherapy of tumors. World J Surg 1983;7:700-709. [2]

Hayata Y, Kato H. Gan To Kagaku Ryoho. Laser and cancer therapy. Cancer Chemother 1983;10:1387-94.

[3]

Kabat-Zinn J, Wheeler E, Light T, Skillings A, Scharf MJ, Cropley TG, et al. Influence of a mindfulness meditation-based stress reduction intervention on rates of skin clearing in patients (PUVA). Psychosom Med 1998;60:625-632.

[4]

RI PT

with moderate to severe psoriasis undergoing photo therapy (UVB) and photochemotherapy Parrish JA, Jaenicke KF. Action spectrum for phototherapy of psoriasis. J Investig Dermatol 1981;76:359-362.

[5] Gobin AM, Lee MH, Halas NJ, James WD, Drezek RA, West JL. Near-infrared resonant nanoshells for

SC

combined optical imaging and photothermal cancer therapy. Nano Lett 2007;7:1929-1934.

[6] Loo C, Lowery A, Halas N, West J, Drezek R. Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Lett 2005;5:709-711.

M AN U

[7] Huang X, El-Sayed IH, Qian W, El-Sayed MA. Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. J Am Chem Soc 2006;128:2115-2120. [8] Bonnett R. Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy. Chem Soc Rev 1995;24:19-33.

[9] Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, et al. Photodynamic therapy. J Natl Cancer Inst 1998;90:889-905.

Dolmans DEJGJ, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer 2003;3:380-387.

TE D

[10]

[11] Gao L, Fei J, Zhao J, Li H, Cui Y, Li J. Hypocrellin-loaded gold nanocages with high two-photon efficiency for photothermal/photodynamic cancer therapy in vitro. ACS Nano 2012;6:8030-8040. [12]

Kelly KM, Kimel S, Smith T, Stacy A, Hammer-Wilson MJ, Svaasand LO, et al. Combined

EP

photodynamic and photothermal induced injury enhances damage to in vivo model blood vessels. Lasers Surg Med 2004;34:407-413.

AC C

[13] Kah JCY, Wan RCY, Wong KY, Mhaisalkar S, Sheppard CJR, Olivo M. Combinatorial treatment of photothermal therapy using gold nanoshells with conventional photodynamic therapy to improve treatment efficacy: An in vitro study. Lasers Surg Med 2008;40:584-589.

[14] Kuo W-S, Chang Y-T, Cho K-C, Chiu K-C, Lien C-H, Yeh C-S, et al. Gold nanomaterials conjugated with indocyanine green for dual-modality photodynamic and photothermal therapy. Biomaterials 2012;33:3270-3278.

[15] Jang B, Park J-Y, Tung C-H, Kim I-H, Choi Y. Gold nanorod−photosensitizer complex for nearinfrared fluorescence imaging and photodynamic/photothermal therapy in vivo. ACS Nano 2011;5:1086-1094. [16] Kuo W-S, Chang C-N, Chang Y-T, Yang M-H, Chien Y-H, Chen S-J, et al. Gold nanorods in photodynamic therapy, as hyperthermia agents, and in near-infrared optical imaging. Angew Chem 2010;122:2771-2775.

26

ACCEPTED MANUSCRIPT [17] Lin J, Wang S, Huang P, Wang Z, Chen S, Niu G, et al. Photosensitizer-loaded gold vesicles with strong plasmonic coupling effect for imaging-guided photothermal/photodynamic therapy. ACS Nano 2013;7:5320-5329. [18] Wang S, Huang P, Nie L, Xing R, Liu D, Wang Z, et al. Single continuous wave laser induced photodynamic/plasmonic photothermal therapy using photosensitizer-functionalized gold nanostars. Adv Mater 2013;25:3055-3061.

RI PT

[19] Murakami T, Nakatsuji H, Inada M, Matoba Y, Umeyama T, Tsujimoto M, et al. Photodynamic and photothermal effects of semiconducting and metallic-enriched single-walled carbon nanotubes. J Am Chem Soc 2012;134:17862-17865.

[20] Sahu A, Choi WI, Lee JH, Tae G. Graphene oxide mediated delivery of methylene blue for combined photodynamic and photothermal therapy. Biomaterials 2013;34:6239-6248. by nano-graphene oxide. ACS Nano 2011;5:7000-7009.

SC

[21] Tian B, Wang C, Zhang S, Feng L, Liu Z. Photothermally enhanced photodynamic therapy delivered [22] Peng J, Zhao L, Zhu X, Sun Y, Feng W, Gao Y, et al. Hollow silica nanoparticles loaded with

M AN U

hydrophobic phthalocyanine for near-infrared photodynamic and photothermal combination therapy. Biomaterials 2013;34:7905-7912.

[23] Khlebtsov B, Panfilova E, Khanadeev V, Bibikova O, Terentyuk G, Ivanov A, et al. Nanocomposites containing

silica-coated

gold–silver

nanocages

and

Yb–2,4-dimethoxyhematoporphyrin:

multifunctional capability of IR-luminescence detection, photosensitization, and photothermolysis. ACS Nano 2011;5:7077-7089.

[24] Rothschild BM. Thermographic assessment of bone and joint disease. Orthop Rev 1986;15:765-80.

TE D

[25] Ring EF, Ammer K. Infrared thermal imaging in medicine. Physiol Meas 2012;33:R33-46. [26] Lahiri BB, Bagavathiappan S, Jayakumar T, Philip J. Medical applications of infrared thermography: A review. Infrared Phys Techn 2012;55:221-235. [27] Karpman HL, Knebel A, Semel CJ, Cooper J. Clinical studies in thermography. II. Application of

EP

thermography in evaluating musculoligamentous injuries of the spine--a preliminary report. Arch Environ Health 1970;20:412-7.

[28] Wang LV, Hu S. Photoacoustic tomography: in vivo imaging from organelles to organs. Science

AC C

2012;335:1458-1462.

[29] Beard P. Biomedical photoacoustic imaging. Interface Focus 2011;1:602-631. [30] Xu M, Wang LV. Photoacoustic imaging in biomedicine. Revi Sci Instrum 2006;77:041101-22. [31] Wang X, Pang Y, Ku G, Xie X, Stoica G, Wang LV. Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain. Nat Biotechnol 2003;21:803-806. [32] Xiang L, Wang B, Ji L, Jiang H. 4-D Photoacoustic tomography. Sci Rep 2013;3:1113-1120. [33] Wilson K, Homan K, Emelianov S. Biomedical photoacoustics beyond thermal expansion using triggered nanodroplet vaporization for contrast-enhanced imaging. Nat Commun 2012;3:618-627. [34] De La Zerda A, Zavaleta C, Keren S, Vaithilingam S, Bodapati S, Liu Z, et al. Carbon nanotubes as photoacoustic molecular imaging agents in living mice. Nat Nanothechnol 2008;3:557-562. [35] Maji SK, Sreejith S, Joseph J, Lin M, He T, Tong Y, et al. Upconversion nanoparticles as a contrast agent for photoacoustic imaging in live mice. Adv Mater 2014;26:5633-5638.

27

ACCEPTED MANUSCRIPT [36] Song W, Zheng W, Xu Q, Lin R, Gong X, Song L. Reflection-mode subwavelength-resolution photoacoustic microscopy for label-freemicrovascular imaging in vivo. in optics in the life sciences. OSA Technical Digest (online) (Optical Society of America, 2015), paper BrT2B.4. [37] Yao J, Wang L, Yang J-M, Maslov KI, Wong TTW, Li L, et al. High-speed label-free functional photoacoustic microscopy of mouse brain in action. Nat Methods 2015;12:407-410. [38] Chen X, Liu L, Liu Z, Marcus MA, Wang WC, Oyler NA, et al. Properties of disorder-engineered [39]

RI PT

black titanium dioxide nanoparticles through hydrogenation. Sci Rep 2013;3:1-7. Chen X, Liu L, Yu PY, Mao SS. Increasing solar absorption for photocatalysis with black hydrogenated titanium dioxide nanocrystals. Science 2011;331:746-750.

[40] Lin T, Yang C, Wang Z, Yin H, Lu X, Huang F, et al. Effective nonmetal incorporation in black titania with enhanced solar energy utilization. Energy Environ Sci 2014;7:967-972.

SC

[41] Yang C, Wang Z, Lin T, Yin H, Lü X, Wan D, et al. Core-shell nanostructured “black” rutile titania as excellent catalyst for hydrogen production enhanced by sulfur doping. J Am Chem Soc 2013;135:17831-17838.

M AN U

[42] Wang Z, Yang C, Lin T, Yin H, Chen P, Wan D, et al. Visible-light photocatalytic, solar thermal and photoelectrochemical properties of aluminium-reduced black titania. Energy Environ Sci 2013;6:3007-3014.

[43] Ren W, Yan Y, Zeng L, Shi Z, Gong A, Schaaf P, et al. A near infrared light triggered hydrogenated black TiO2 for cancer photothermal therapy. Adv Healthcare Mater 2015:n/a-n/a. [44] Murakami Y, Kenji E, Nosaka AY, Nosaka Y. Direct detection of OH radicals diffused to the gas phase from the UV-Irradiated photocatalytic TiO2 surfaces by means of laser-induced fluorescence

TE D

spectroscopy. J Phys Chem B 2006;110:16808-16811. [45] Nosaka Y, Nakamura M, Hirakawa T. Behavior of superoxide radicals formed on TiO2 powder photocatalysts studied by a chemiluminescent probe method. Phys Chem Chem Phys 2002;4:10881092.

Kubo W, Tatsuma T. Detection of H2O2 released from TiO2 photocatalyst to air. Anal Sci

EP

[46]

2004;20:591-3.

[47] Miyoshi N, Kume K, Tsutumi K, Fukunaga Y, Ito S, Imamura Y, et al. Application of titanium

AC C

dioxide (TiO2) nanoparticles in photodynamic therapy (PDT) of an experimental tumor. AIP Conf Proc 2011;1415:21-23.

[48] Yamaguchi S, Kobayashi H, Narita T, Kanehira K, Sonezaki S, Kubota Y, et al. Novel photodynamic therapy using water-dispersed TiO2-polyethylene glycol compound: evaluation of antitumor effect

on glioma cells and spheroids in vitro. Photochem Photobiol 2010;86:964-71.

[49] Zhang H, Shan Y, Dong L. A comparison of TiO2 and ZnO nanoparticles as photosensitizers in photodynamic therapy for cancer. J Biomed Nanotechnol 2014;10:1450-7. [50] Li J, Wang X, Jiang H, Lu X, Zhu Y, Chen B. New strategy of photodynamic treatment of TiO2 nanofibers combined with celastrol for HepG2 proliferation in vitro. Nanoscale 2011;3:3115-3122. [51] Wang Z, Yang C, Lin T, Yin H, Chen P, Wan D, et al. H-doped black titania with very high solar absorption and excellent photocatalysis enhanced by localized surface plasmon resonance. Adv Funct Mater 2013;23:5444-5450.

28

ACCEPTED MANUSCRIPT [52]

Ma X, Dai Y, Yu L, Huang B. Noble-metal-free plasmonic photocatalyst: hydrogen doped semiconductors. Sci Rep 2014;4:3986-3993.

[53] Yang G, Jiang Z, Shi H, Xiao T, Yan Z. Preparation of highly visible-light active N-doped TiO2 photocatalyst. J Mater Chem 2010;20:5301-5309. [54] König K. Multiphoton microscopy in life sciences. J Microsc 2000;200:83-104. [55] Ohyashiki T, Nunomura M, Katoh T. Detection of superoxide anion radical in phospholipid liposomal Biophys Acta (BBA)-Biomembr 1999;1421:131-139. [56]

RI PT

membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran. Biochimica Carloni P, Damiani E, Greci L, Stipa P, Tanfani F, Tartaglini E, et al. On the use of 1,3diphenylisobenzofuran (DPBF). Reactions with carbon and oxygen centered radicals in model and natural systems. Res Chem Intermed 1993;19:395-405.

SC

[57] Wang J, Tang HY, Yang WL, Chen JY. Aluminum phthalocyanine and gold nanorod conjugates: the combination of photodynamic therapy and photothermal therapy to kill cancer cells. J Porphyrins Phthalocyanines 2012;16:802-808.

M AN U

[58] Spiller W, Kliesch H, WÖhrle D, Hackbarth S, RÖder B, Schnurpfeil G. Singlet oxygen quantum yields of different photosensitizers in polar solvents and micellar solutions. J Porphyrins Phthalocyanines 1998;02:145-158.

[59] Wang Z, Ma W, Chen C, Ji H, Zhao J. Probing paramagnetic species in titania-based heterogeneous photocatalysis by electron spin resonance (ESR) spectroscopy—A mini review. Chem Eng J 2011;170:353-362. [60]

Lang K, Wagnerová DM, Stopka P, Damerau W. Reduction of dioxygen to superoxide

TE D

photosensitized by anthraquinone-2-sulphonate. J Photochem Photobiol A 1992;67:187-195. [61] Arjomandnejad M, Muhammadnejad A, Haddadi M, Sherkat-Khameneh N, Rismanchi S, Amanpour S, et al. HeLa cell line xenograft tumor as a suitable cervical cancer model: growth kinetic characterization and immunohistochemistry array. Arch Iran Med 2014;17:273-7.

EP

[62] Yin ZF, Wu L, Yang HG, Su YH. Recent progress in biomedical applications of titanium dioxide. Phys Chem Chem Phys 2013;15:4844-58. [63] Shi H, Magaye R, Castranova V, Zhao J. Titanium dioxide nanoparticles: a review of current

AC C

toxicological data. Part Fibre Toxicol 2013;10:15.

[64] Skocaj M, Filipic M, Petkovic J, Novak S. Titanium dioxide in our everyday life; is it safe? Radiol Oncol 2011;45:227-247.

[65] Liu X, Ren X, Deng X, Huo Y, Xie J, Huang H, et al. A protein interaction network for the analysis of the neuronal differentiation of neural stem cells in response to titanium dioxide nanoparticles. Biomaterials 2010;31:3063-70. [66] Chen Q, Chen H, Shapiro H, Hetzel FW. Sequencing of combined hyperthermia and photodynamic therapy. Radiat Res 1996;146:293-7. [67] Gottfried V, Kimel S. Temperature effects on photosensitized processes. J Photochem Photobiol B 1991;8:419-30.

29

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT