TiO2âx Based Nanoplatform for Bimodal Cancer Imaging and NIR

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TiO Based Nano-platform for Bimodal Cancer Imaging and NIRTriggered Chem/Photodynamic/Photothermal Combination Therapy Wei Guo, Fei Wang, Dandan Ding, Chuanqi Song, Chongshen Guo, and Shaoqin Liu Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b03241 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 21, 2017

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Chemistry of Materials

TiO2-x Based Nano-platform for Bimodal Cancer Imaging and NIR-Triggered Chem/Photodynamic/Photothermal Combination Therapy Wei Guo, ‡a,b Fei Wang, ‡a,b Dandan Ding,b Chuanqi Song,b Chongshen Guo,* a,b Shaoqin Liu* a,b a

School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.

b

Key Lab of Microsystem and Microstructure (Ministry of Education), Academy of Fundmental and Interdisciplinary Sciences, Harbin Institute of Technology, Harbin 150080, China.

‡ Equal

contribution from Wei Guo and Fei Wang

Corresponding author: [email protected] (C. S. Guo)

[email protected] (S. Q. Liu)

KEYWORDS: Theranostics, Photothermal therapy, Photodynamic therapy, Fluorescence imaging, Photoacoustic imaging

ACS Paragon Plus Environment

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ABSTRACT

Integration of cancer diagnosis and treatment, namely theranostics, is an important issue in biomedical field. Benefiting from excellent photothermal effect, ROS generation ability and desired mesoporous structure of TiO2-x matrix, we strategically designed and fabricated TiO2-x based theranostic system for realizing fluorescence/photoacoustic tomography (PAT) bimodal imaging guided triple-therapy of photothemal/photodynamic/chemotherapy in this work. Nonstoichiometric TiO2-x nanospheres are excellent near-infrared absorptive material, which takes on both photosensitizer and photothermal agent roles in implementing PDT/PTT combination therapy and PAT imaging. Moreover, mesoporous structure of TiO2-x also allowed drug loading and polydopamine sealing layer enabled it to induce NIR/pH-triggered drug controlled release. Resultantly, both of in vitro and in vivo experiment manifested the remarkably tumor inhibition and tumor imaging effects by TiO2-x based theranostic system. The antitumor mechanism was attributable to a synergistic therapeutic effect (Combination index = 0.318) of DOX-induced DNA damage, and PDT/PTT caused mitochondrial dysfunction and change of cell membrane permeability. Innovatively, the B-mode ultrasonography was adopted to monitor the rehabilitation process at solid tumor site after treatment, which observed a liquefaction necrosis process.


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INTRODUCTION Significant side effects, inevitable secondary actions and poor therapeutic efficacies from traditional antitumor therapies (like radiotherapy or chemotherapy) in clinical practice motivate medical scientists to seek for a new way that is expected to achieve better treatment outcome, high accuracy of therapy and great controllability.1-5 The first representative strategy is controlled drug delivery and releasing process which requires fewer dosage of drug in virtue of on-site and real-time releasing, hence, it shows great superiority than traditional chemotherapy and results in less side effect.6-8 Meanwhile, near-infrared-mediated phototherapy is also regarded as a promising alternative to radiotherapy on the merits of low body damage.9-11 Phototherapy relies on light-triggered local hyperthemia (photothermal therapy, PTT) or reactive oxygen species (ROS, photodynamic therapy, PDT) to realize high spatiotemporal accuracy since the antitumor effect only takes place at site where both incident light and optical active substance are available.12,13 Despite all this, above three emerging therapeutic methods are still in the developing stage and any of them alone can hardly generate desired tumor treatment effect. Even by controlled drug delivery, prolonged administration of antitumor drug still causes the drug resistance and leads to deterioration in treatment outcome. PDT mainly takes effect in the early stage of treatment and its efficiency will remarkably decrease with the oxygen depletion around tumor.14-16 While for PTT, the highlighted performance exhibits in the late stage of treatment because it requires a procedure to reach critical temperature for cancer cells killing.17 However, the local hyperthemia can also cause heat shock response and lower the PTT efficiency.18 What is exciting, the combination therapy involves in above two or more therapeutic approaches engenders a significant improvement. For example, collaborative


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methods of PTT/PDT or PTT/drug controlled releasing, have been tentatively investigated and demonstrated the synergetic therapeutic actions in the previous works.19-22 Another approach to promote therapeutical accuracy and reduce damage to healthy tissue is to introduce image surveillance in the process of cancer treatment. Theranostics of imaging-guided tumor treatment can realize real-time regulation.23-26 Among various imaging modalities, photoacoustic tomography imaging (PAT) depends on light-triggered photothermal effect and subsequent ultrasonic waves,27,28 which could be synchronously implemented with PDT/PTT under

NIR

irradiation

and

is

more

compatible

with

NIR-triggered

chem/photodynamic/photothermal combination therapy. The hydrogenated black titanium dioxide nanoparticles have drew much attentions from researchers of different areas since chen et al. reported them in 2011.29 However, very few efforts worked on employing TiO2-x for the cancer therapy until now.30-32 In this work, we established a TiO2-x based nano-platform for fluorescence/PAT bimodal cancer imaging and NIR-triggered chem/photodynamic/photothermal combination therapy. The TiO2-x sphere matrix possesses strong optical absorbance in the NIR region and can produce both of hyperthemia and ROS upon NIR irradiation, thus producing triple functions of PTT, PDT and PAT imaging. What is more, the ordered mesoporous channels inside the TiO2-x sphere matrix enable it to be drug carrier after sealing by polydopamine layer, which is pH and photothermal sensitive shell for DOX controlled releasing. Finally, fluorescence cancer imaging could be achieved as fluorescent molecular of Cy5.5 labeling on the outer layer of polydopamine (Schematic 1). Herein, TiO2-x matrix behaves as both photosensitizer (PS) for PDT and photothermal agent (PTA) for PTT, which could exempt from problem of using different ingredients for PS and PTA in other’s work,


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such as mismatch of absorption band and mutual interference between PS and PTA, as well as operation complexity.

Schematic 1. Schematic illustration of synthetic method and bioimaging-guided triple-therapy. EXPERIMENTAL SECTION Materials. Titanium (IV) isopropoxide, Calcein-AM, propidium iodide (PI), dopamine, 3-[4,5dimethylthialzol-2-yl]-2,5-diphenyltetrazolium

bromide

(MTT),

1,3-diphenylisobenzofuran

(DPBF) and 2’,7’-dichlorodihydrofluoresceindiacetate (H2DCFDA) were obtained from SigmaAldrich. Doxorubicin hydrochloride (DOX), 4',6-diamidino-2-phenylindole (DAPI), ethanol and JC-1 were purchased from Aladdin. Cy5.5 was obtained from Shanghai Jun Sheng Biotechnology Co., Ltd. Synthesis of TiO2 and TiO2-x Nanospheres. TiO2 nanospheres were prepared by a solvothermal method. Firstly, 0.1 g of titanium isopropoxide was dissolved into the 45 mL ethanol with magnetic stirring, forming a clear solution. Then, 5 mL of acetic acid was added into above solution. The resulting solution was transferred into a Teflon-lined autoclave of 100


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mL internal volume and heated at 200 °C for 4 h. After the reaction, the white powder was centrifuged, washed with water and ethanol. Then, sample was collected after vacuum drying at 60 oC. The TiO2-x nanosphere was synthesized by reducing above TiO2 powder at 500 oC for 1 h under hydrogen atmosphere. DOX loading and Cy 5.5 modification. Firstly, 5 mg of TiO2-x was dissolved into the 5 mL Tris-HCl solution (1 M, pH = 8.5) under ultrasonic dispersion. Then, 1 mL DOX solution (1 mg/mL in Tris-HCl) was added into above solution. The mixture was stirred at room temperature for 12 h. After that, 5 mL of dopamine in Tris-HCl solution (2 mg/mL) was added. After stirring for 2 h, the product was centrifuged and washed with DI water. Then, the content of DOX in the supernatant was measured using fluorescence spectrometer (Ex: 495 nm, Em: 595 nm). The experiment was repeated for three times. The loading efficiency (LE) of DOX was calculated according to the following equation. LE(%)=

Mt-Mu ×100% Mt

where Mt is the total mass of DOX for drug loading, Mu is the mass of unencapsulated DOX. The drug loaded solid powder of DOX@TiO2-x@PDA was obtained by freeze-drying. Afterwards, 12 mg of DOX@TiO2-x@PDA was dissolved in the 20 mL DI water under ultrasonic dispersion. Then, 8 mg of EDC and 12 mg of NHS were added into above solution with magnetic stirring for 0.5 h. 1.6 mg of Cy 5.5 was subsequently introduced into the mixed solution with magnetic stirring for another 12 h. The final product of DOX@[email protected] was collected after freeze-drying. DOX releasing test. The drug release profiles of DOX were studied at 37.5 °C, pH 5.5 or 7.4, in phosphate buffer solution. Briefly, 10 mg of DOX@[email protected] was dispersed in 5 mL of PBS solution and sealed in a dialysis bag (molecular weight cutoff 8000), which was


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submerged in 20 mL of PBS solution later. At selected intervals, the solution was taken out to determine the release amount by fluorescence spectrometer. The drug release of DOX from the DOX@[email protected] under NIR irradiation (1.0 W/cm2) was performed by a similar way. Characterization. Transmission electron microscopy (TEM) images were obtained on a JEM1400 microscope at an acceleration voltage of 100 kV. The phase composition of the sample was determined by X-ray diffraction analysis (XRD, Shimadzu XD-D1). The chemical valence of Ti ions was measured by X-ray photoelectron spectroscopy (XPS, Perkin Elmer PHI 5600). The optical properties were measured using a spectrophotometer (U-4100. Hitachi). The thermogravimetric analysis (TG, Rigaku, TG8101D) was performed from room temperature to 800 °C with a heating rate of 10 °C/min in the air. MTT experiments were measured using a microplate reader (Infinite M200, Tecan). B-mode ultrasonography experiments performed on a HI VISION Ascendus system. Cell Culture. MDA-MB-231 cells were grown in monolayer in DMEM. They were all supplemented with 10% (v/v) fetal bovine serum (FBS, Gibbico) and penicillin/streptomycin (100 U mL-1 and 100 mg mL-1, respectively, Gibco) in a humidified 5% CO2 atmosphere at 37 °C. Detection of ROS. The extracellular ROS generation was divided into two groups, that are, pure water with NIR 808 nm irradiation and TiO2-x solution (500 μg/mL) with NIR 808 nm irradiation. The DPBF was employed to detect the ROS generation. Briefly, 20 μL of N,Ndimethylformamide solution containing DPBF (1 mg/mL) was added to 3 mL of above two solutions. After that, they were irradiated by NIR laser for varied durations. After centrifugation, the collected supernatant was tested by a spectrophotometer. MDA-MB-231 cell lines were chose to detect intracellular ROS level by staining with the H2DCFDA probe. 5 × 103 cells per


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well were seeded into a 6-well plate and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 h. MDA-MB-231 cells were incubated with 200 μL TiO2-x@PDA (250 μg/mL) at 37 °C for 24 h. The positive control cells were prepared with 200 μL 50 mM H2O2 at 37 °C for 30 min. Cells were incubated with 50 μL H2DCFDA (10 mM in DMSO) for another 1 h at 37 °C. After that, cells were washed with PBS twice and irradiated under the 808 nm laser (1.0 W/cm2) for 10 min. ROS level was immediately measured by Olympus BX53 fluorescence microscope using an excitation of 488 nm and an emission of 515-540 nm. In vitro and In vivo PA imaging. The in vitro and in vivo PA imaging experiments were performed on a MOST invision 128 system. Briefly, 1 mL of the DOX@[email protected] aqueous at various concentrations (0, 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL) was added into the agar-phantom container and placed in the testing system for signal detection in vitro. The in vivo PAT imaging of DOX@[email protected] was carried out on MDA-MB-231 tumorbearing mice. The PAT imaging data of tumor site were collected before and after the intratumoral injection of DOX@[email protected] solution (100 μL, 2 mg/mL). Cytotoxity assay (MTT). The viability of cells was investigated using an MTT assay. For in vitro PDT experiment, MDA-MB-231 cells seeded in 96-well plate were incubated with TiO2x@PDA

(250 μg/mL) for 24 h, and then irradiated by the 808 nm laser at a power density of 1.0

W/cm2 on an ice box for 10 min. Whereas for in vitro PTT experiments, 50 μL of sodium azide (10 μM) was added into the cells which had been incubated with TiO2-x@PDA (250 μg/mL) for 24 h, and then irradiated by the 808 nm laser at a power density of 1.0 W/cm2 for 10 min. In vitro combination of PDT/PTT experiment was carried out in same way to above, but it is free of ice bath and addition of sodium azide. For triple therapy of PDT/PTT/chemotherapy, DOX@TiO2x@PDA

and 10 min 808 nm laser irradiation were applied in a similar way to above. Finally, a


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typical MTT assay was used to determine the relative cell viabilities. The assay was performed out in triplicate. In brief, resultant cells were incubated in media containing 20 μL MTT (5 mg/mL) for 4 h. Then, the media with MTT were removed, and 150 μL of DMSO was added to dissolve formazan crystal at room temperature for 30 min. The absorbance was measured at 490 nm by multi-detection microplate reader (Synergy TMHT, BioTek Instruments Inc., USA). In vitro living-dead staining. MDA-MB-231 cells were incubated in a 35 mm quartz cuvette at a density of 3 x 105 cells per dish and were allowed to grow for 24 h at 37 °C. Then, 2.0 mL of medium containing the TiO2-x@PDA or DOX@TiO2-x@PDA (250 μg/mL) was added into the cuvette to replace the culture medium. After incubated for another 2 h, the cells were rinsed three times with PBS. After that, the cells were irradiated with 808 nm laser for different time intervals (2, 6, and 10 min, 1.0 W/cm2). Finally, the irradiated cells were rinsed with PBS and stained with calcein-AM and PI, respectively. The stained cells were immediately measured by Olympus BX53 fluorescence microscope. Mitochondrial membrane potential change. MDA-MB-231 cells were cultured with TiO2x@PDA

solution (250 μg/mL) in DMEM culture medium for 2 h. Then, the cells were rinsed

three times with PBS. Afterwards, the cells were irradiated under 808 nm laser (1.0 W/cm2) for 10 min. Cells treated with TiO2-x@PDA solution without irradiation were used as control. Subsequently, the medium was removed and JC-1 staining solution was added according to the manufacturer protocol. After staining for 25 min, cells were washed and imaged by Olympus BX53 fluorescence microscope. In vivo antitumor effect. Female BALB/C nude mice (5 weeks old) were obtained from vitalriver experimental animal technical co., LTD (Beijing) and all the in vivo experiments were implemented according to the criterions of the National Regulation of China for Care and Use of


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Laboratory Animals. Firstly, the tumors were inoculated by subcutaneous injection of 1×107 MDA-MB-231 cells in the left flank of each BALB/c mouse using 50 % matrigel. After grown for about two weeks, the tumor size reached about 200 mm3. The tumor-bearing nude mice were randomized into five groups (n = 5, each group), (1) control group received 100 μL of PBS injection only; (2) group with 100 μL (1 mg/mL) of TiO2-x@PDA injection; (3) group with NIR irradiation; (4) group with both of NIR irradiation and TiO2-x@PDA; (5) group with both of NIR laser and DOX@[email protected] group. The power density of NIR laser was 1.0 W/cm2, and the treatment began at 2 h post intratumorally injection. The irradiation duration for groups with NIR irradiation is 10 min. The tumor inhibition effects were evaluated by measuring the tumor volumes, which is calculated as tumor volumes (V) = length × width2/2. Relative tumor volume was calculated as V/V0 (V0 was the tumor volume before treatment). Relative body weight was calculated as W/W0 (W0 was the corresponding mouse’s weight when the treatment was initiated). The combination of among chemotherapy, PDT and PTT was evaluated by Chou, T.-C. and Martin, N. CompuSyn software for drug combinations and for general dose effect analysis, and user’s guide (ComboSyn, Inc. Paramus, NJ 2007). The CI value quantitatively defines synergism (CI1).33-35 Histology analyses. Histology analysis was carried out at the 14th day after different treatments. The major organs and tumor tissues were isolated, fixed in 4% paraformaldehyde solution and embedded in paraffin. The sliced tumor tissues and organs were stained with hematoxylin and eosin (H&E) and analyzed by Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan).


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RESULTS AND DISCUSSION Firstly, we synthesized the mesoporous TiO2 nanospheres by a solvothermal method according to our previous work.36 The diameter of these uniform TiO2 sphere is in the range of 300-500 nm (Figure 1a & Figure S1 and S2). Then, the non-stoichiometric TiO2-x was obtained by reducing above TiO2 nanospheres under hydrogen atmosphere at 500 oC for 1 h. As can be seen in Figure 1b and S2, the TiO2-x NPs remain the spherical shape and good dispersion compared with the TiO2 after a hydrogen reduced process. In addition, they possess nearly the same hydrodynamic diameter (TiO2: 599.9 nm, TiO2-x: 606.0 nm). Therefore, the TiO2-x crystal well inherits the size and morphological characteristics of TiO2 nanospheres after reduction. Then, a typical anticancer drug of doxorubicin hydrochloride (DOX) was encapsulated in the mesoporous pores of TiO2-x (named as DOX@TiO2-x) and followed by PDA modification onto the surface of DOX@TiO2-x by a well-established method to form a DOX@TiO2-x@PDA structure,37 which could avoid premature drug release and achieve a controlled drug release. The final procedure of binding Cy5.5 fluorescent molecular to PDA layer was realized by a standard EDC/NHS method (Schematic 1). From Figure 1c, we can see that the thickness of the PDA/Cy5.5 layer is about 31 nm in final sample of DOX@[email protected]. The N2 adsorption-desorption isotherm shown in Figure 1d reveals that both of the TiO2 and TiO2-x isotherms are of typical IV curve with hysteresis loops locating at relative pressure (P/P0) of 0.4-0.7, indicating that the capillary condensation happened in the mesoporous pores. After reduction process, the specific surface area decreased from 142.27 m2/g of TiO2 to 84.43 m2/g for TiO2-x, but the area of hysteresis loop became larger. In addition, there is no difference on the pore size for TiO2 and TiO2-x, both of which are around 3.5 nm. For the DOX@[email protected], it has very low specific surface area (5.06 m2/g) and no porous structure characteristic, suggesting the successful DOX loading


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and PDA modification (Figure1 d, e). The crystal phase of the obtained TiO2, TiO2-x, DOX@[email protected] could be well indexed to anatase (JCPDS NO. 84-1285),

Figure 1. Characterization of samples. (a-c) TEM images of TiO2, TiO2-x and DOX@[email protected],

respectively. (d, e) Nitrogen adsorption-desorption isotherms and pore size

distribution, (f) XRD patterns, (g) Fitted Ti 2p XPS spectra of TiO2-x, (h) FT-IR spectra, (i) Thermogravimetric curve (TG). illustrating that the reduction process, drug loading and surface modification processes have limited influences on crystallographic structure (Figure 1f). However, we suspected that the


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surface oxygen defects maybe emerged after reduction process since we observed an obvious color change from white to dark gray.Thus, X-ray photoelectron spectroscopy (XPS) survey was employed to confirm the chemical valence of Ti ions in reduced sample. As expected, there are two spin-orbit doublets at Ti2P 3/2 458.9 and Ti2P 1/2 464.6 eV, and Ti2P 3/2 457.8 and Ti2P 1/2 463.5 eV in the Figure 1g, corresponding to Ti4+ and Ti3+ ions, respectively. Hence, XPS result verified the composition of TiO2-x and this is consistent with previous report.38,39 Then, the FTIR spectroscopy was used to confirm the success in establishing DOX@[email protected] structure. As illustrated in Figure 1h, the TiO2-x only shows a broad band at 587 cm-1 relating to the Ti-O mode. The absorption peaks at 2956 and 2925 cm-1, 1386 cm-1 are assigned to the C-H stretching and symmetric bending modes of PDA layer. In addition, it also gives the characteristic absorption peaks at 1597 and 1386 cm−1 due to the polymeric structure. Besides characteristic peaks of TiO2-x and PDA, the DOX@[email protected] also possesses phenolic hydroxyl (1215 cm−1), alcohol hydroxyl (1115 cm−1) and C=N (1457 cm−1) from DOX and Cy5.5, thus confirming the achievement of drug loading, PDA coating and Cy5.5 modification. Finally, thermogravimetric (TG) measurement was used to determine the contents of PDA layer and DOX roughly. As shown in Figure 1i, the weight loss difference between TiO2-x and TiO2x@PDA

originates from the PDA modification, which indicates that the PDA layer accounts for

6.4 wt% in TiO2-x @PDA. Considering the trace amount of Cy5.5, the further weight loss of DOX@TiO2-x@PDA-Cy 5.5 is mainly because of DOX decomposition. By this way, about 12.04 wt% DOX is determined to be loaded in TiO2-x mesopore. Then,

we

checked

the

photoabsorption

of

powder

samples

using

UV-vis-NIR

spectrophotometer. Compared with white TiO2 that shows limited optical absorption in biological window of 800-1300 nm, the non-stoichiometric TiO2-x displays higher absorption in


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the NIR region of 800 to 1500 nm and the absorbance at 808 nm is about 4-fold of TiO2, making it to be a splendid NIR absorbing material (Figure 2a). The absorption bands at 500 nm and 678 nm

Figure 2. Photoabsorption and photothermal effect. (a) UV-vis-NIR spectra of powder TiO2, TiO2-x, TiO2-x@PDA and DOX@[email protected]. (The upside shows the color of samples) (b) UV-vis-NIR spectra of DOX@[email protected] solution with different concentrations. (c) The photothermal heating curves of DOX@[email protected] solutions with different concentrations under NIR irradiation at a power density of 1.0 W/cm2. (d) Plot of temperature change (ΔT) over a period of 10 min irradiation versus DOX@[email protected] concentration.


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are characteristic absorption of DOX and Cy5.5 in DOX@[email protected], suggesting successful drug-loading and fluorescence labeling again. In addition, integration of PDA, DOX and Cy5.5 to the TiO2-x matrix also enhances the optical absorption in the NIR range slightly, which is desirable for NIR-triggered phototherapy. The optical response of DOX@[email protected]

solutions with varied concentration was also investigated. As can be seen in

Figure 2b, the absorbance of the DOX@[email protected] solution remarkably enhanced with its concentration increment. To evaluate the photothermal effect of DOX@[email protected], we next measured temperature rise of DOX@[email protected] solution under 808 nm NIR laser (1.0 W/cm2) irradiation. In Figure 2 c & d, the temperature of solutions increased with concentration of DOX@[email protected] and NIR irradiation duration, proving the role of DOX@[email protected] nano-platform as photothermal agent. The effective ROS generation is necessary requirement of a photosensitizer for PDT implement. We next investigated the photosensitive ability of TiO2-x to form ROS. The 1,3-diphenylisobenzofuran (DPBF) and 2’,7’dichlorofluorescin diacetate (H2DCFDA) probes were employed to detect the extracellular and intracellular ROS production, respectively.40 DPBF probe for detecting extracellular ROS production lies in its decomposition by ROS. Resultantly, a decrement of DPBF characteristic absorption could be observed accordingly. As displayed in Figure 3a, the absorbance of the DPBF solution with TiO2-x decreased significantly as irradiation time went on, suggesting the sufficient ROS level induced by TiO2-x. In contrast, the pure water led to very limited ROS generation with same laser irradiation. The ROS generation was further confirmed by ESR spectra. Firstly, the 5, 5’-dimethylpyrroline-1-oxide (DMPO) was used as spin trap agent for the detection of hydroxyl radicals. After irradiation (808 nm laser, 1.0 W/cm2) for 10 min, the appearance of 1:2:2:1 multiplicity in the ESR spectrum of TiO2-x solution (0.5 mg/mL) can be


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assigned to the characteristics of DMPO-OH adduct, indicating the generation of hydroxyl radicals30 (Figure S3a). Moreover, the generation of singlet oxygen was also detected by ESR method with a trapping agent of 2, 2, 6, 6-tetramethyl-4-piperidone (TEMP). As can be seen in Figure S3b, when the mixture of TiO2-x solution (0.5 mg/mL) and TEMP probe is irradiated by a 808 nm NIR laser (1.0 W/cm2) for 10 min, a typical three-line ESR signal of TEMPO adduct with an equal intensity is obtained, thus suggesting the formation of singlet oxygen.28

Figure 3. ROS generation tests. (a) Absorption spectra of DPBF probe under different irradiation time. (b-e) Fluorescence microscope images of ROS generation in MDA-MB-231 cells after received different treatments: (b) MDA-MB-231 cells control, (c) TiO2-x@PDA treated MDAMB-231 cells, (d) NIR laser treated MDA-MB-231 cells, (e) H2O2 treated MDA-MB-231 cells as a positive control, (f) NIR laser + TiO2-x@PDA treated MDA-MB-231 cells (NIR laser: 1.0 W/cm2, t = 10 min, scale bar = 200 μm).


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Then, intracellular ROS production inside MDA-MB-231 cells was examined by H2DCFDA probe, which would convert to green fluorescent molecules after being oxidized by ROS. The bare MDA-MB-231 cells were used as negative control and the H2O2 (50 mM) treated MDAMB-231 cells were used as a positive control. As shown in Figure 3 b-f, there is no observable green fluorescence from the negative control and TiO2-x@PDA treated cells, while very weak signal is from the NIR irradiated group. The strong green fluorescence emitted from both H2O2 and TiO2-x@PDA + NIR laser irradiation treated groups, indicating that TiO2-x@PDA can produce adequate ROS within cells under NIR irradiation and it is a promising candidate for PDT. To confirm whether H2 reduction treatment and DOX/PDA modification have influences on photoactivity of sample, the ROS generation ability and photothermal effect of TiO2, TiO2-x, DOX@TiO2-x@PDA and DOX@[email protected] were investigated. As can be seen in Figure S4, the DOX/PDA/Cy5.5 modification can obviously enhance the photothermal performance of TiO2-x. Meanwhile, the ROS level determined by DPBF probe reveals that there is only slightly decrement on ROS generation of TiO2-x. Moreover, the DOX and the Cy5.5 can give rise to a chemotherapeutic efficacy and a fluorescence imaging for the mutifunctional theranostic system, respectively. The PDA layer can not only achieve the dual pH/NIR laser irradiation-triggered drug release, but also enhance biocompatibility of TiO2-x. Therefore, PDA layer is ligament for this cancer multifunctional theranostic platform. We then evaluated the bio-imaging behaviors of DOX@[email protected] nano-platform using MDA-MB-231-tumor-bearing nude mice as model animal. The fluorescent molecular of Cy5.5 in the nano-platform allows us to monitor bio-distribution of the DOX@[email protected] in the tumor site using in vivo NIR florescence microscopy (Figure 4a). After intratumoral injection, fluorescence signals from the tumor site are clearly observed. The high


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fluorescent region with a red color indicates real-time location of the DOX@[email protected]. Without NIR irradiation, area of the red region (S) is nearly unchanged within 10 min (Figure 4b). However, we speculated that the NIR irradiation during the phototherapy might enhance the diffusion and expansion of DOX@[email protected] at the tumor site, thus improving antitumor effect. As expected, the spread of DOX@[email protected] happened upon NIR irradiation as evidenced by about 4-fold increment on red area after 10 min treatment (Figure 4a, b), which is close related to the thermal diffusion of the particles under the NIR irradiation. Given this, it is also expected that such a process can promote the distribution of DOX in the solid tumor as well and further enhance the therapeutic effect. PAT imaging is an effective diagnostic technique with high spatial resolution, noninvasiveness and deep penetration. Usually, photothermal materials are potential exogenous contrast agent for PAT imaging, especially in the case of that tissue has a weak PAT signal. Inspired by aforementioned photothermal test, DOX@[email protected] was tentatively used as PAT contrast. In Figure 4 c-e, in vitro test reveals that photoacoustic signal increases with the increment of sample concentration (Figure 4 c-d) and a linear relationship is found between signal intensity and concentration (Figure 4 e). Then, in vivo PAT imaging was carried out by intratumoral injection of DOX@[email protected] and monitoring imaging changes at different time intervals post injection. As shown in Figure 4f, the control group without injection of DOX@[email protected] displays no obvious PAT signal, suggesting that the tumor itself is not an endogenous contrast and needs photothernal material for image formation. In contrast, the signals from tumor location enhanced remarkably after introducing DOX@[email protected]. The PAT signals at tumor site reached maximum after 12 h post-injection and it lasted to 24 h, indicating a long-term imaging effect of DOX@[email protected] via intratumoral


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injection. Taken together, the DOX@[email protected] could be potentially applied to bimodal fluorescence/PAT imaging to sketch tumor.

Figure 4. Fluorescence and PAT imaging. (a) In vivo fluorescence imaging of MDA-MB-231 tumor-bearing mice before and after intratumoral injection of DOX@[email protected]. (The upper group without NIR laser irradiation, while the lower group received laser irradiation). (b) The correlative fluorescence signal ratio of the red area (St/S0) versus time. (S0 is area of red


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region pre-injection, St is area of red region after t min post-injection). (c) PAT signal of DOX@[email protected] solution at different concentrations. (d) In vitro PAT images of DOX@[email protected] dispersed in water with different concentration. (e) PAT signal of DOX@[email protected] as a function of concentration under 808 nm excitation. (f) In vivo PAT images of MDA-MB-231 tumor-bearing mice before and after intratumoral injection of DOX@[email protected] NPs for different post time. (Tumor sites are marked with white dashed circles.)

Next, we investigated the in vitro therapeutic effect of composited nano-platform. Considering on that PTT, PDT and chemotherapy all may induce anti-tumor effect, it is very essential to distinguish the contributions from each one. We designed following methods to realize this. Firstly, TiO2-x@PDA enriched MDA-MB-231 cells were irradiated by NIR laser for 10 min in an ice bath so that the temperature of irradiated area never exceeded 10 oC. By this way, the PTT effect was removed and the cancer cell death was mainly induced by PDT effect (dote as PDT in Figure 5a). Secondly, instead of ice bath, free radical quencher of Na3N was added into above system to eliminate ROS generated by TiO2-x@PDA under NIR irradiation. Thus, it only reflects PTT efficiency (dote as PTT in Figure 5a). Thirdly, a combination therapy of PTT and PDT could be realized by above method but free of ice bath and Na3N addition, where both of photothermal effect and ROS could cause cancer cell death (dote as PTT + PDT). Fourthly, equivalent free DOX to DOX@[email protected] was administrated to MDA-MB-231 cells to determine the contribution of chemotherapy (dote as DOX). Finally, DOX@TiO2-x@PDA was used to simultaneously trigger PTT, PDT and chemotherapy effects on cancer cells under NIR irradiation. MTT assay was carried out for all of above experiments to evaluate the cytotoxicity.


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Figure 5. (a) Relative cell viability of MDA-MB-231 and (b) In vitro living-dead staining studies after different treatments. (Scale bar = 500 μm)

As shown in Figure 5a, for a single function of PDT, PTT or chemotherapy group, the inhibition on MDA-MB-231 cancer cells are just 27.5%, 44.7% and 59.7%, respectively. The inhibition rate on cancer cell could be remarkably enhanced by a combination of PDT and PTT (69.7%), which makes a “dual-punch” effect on killing cancer cells. Obviously, the triple-therapy realized the best treatment effect as expected and it can inhibit 94.8% of MDA-MB-231 cancer cells. On the basis of above results, the combination index is determined to be 0.318. Hence, we can


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conclude that multiple treatments established in this work would be mutual promotion and achieve a synergistic antitumoral effect. Then, we evaluated anti-tumor performance by a living-dead staining. Calcein AM with green fluorescent and propidium iodide (PI) with red fluorescent were used to distinguish the living and dead cells, respectively. In the Figure 5b, the untreated cells, NIR laser treated group and TiO2-x@PDA treated group showed strong green fluorescence but no red fluorescence, indicating that either TiO2-x@PDA or a NIR laser alone caused almost no cells death. For the DOX treated group, there is no apparent red color in the fluorography, but we do find the detachment of death cells from culture dish during the observation. Hence, cell density in the DOX treated group is lower than others due to the departure of DOX-treated death cells. Synergistic action of PTT and PDT realized by applying TiO2-x@PDA and NIR irradiation can give rise to distinct apoptosis and the area of red circle enlarged with irradiation duration. If DOX is loaded in TiO2-x@PDA, additional more massive cells shedding from boundary of light spot to outside were found, (marked by blue arrows), suggesting that the DOX@TiO2-x@PDA is an efficient multifunctional agent with PDT, PTT and chemotherapy effects. The pH-sensitive and NIR irradiation triggered drug release behavior of the DOX@[email protected]

were investigated. The encapsulation efficiency of DOX was 83.5% ± 0.7%

according to the fluorescence spectra result (Figure S5). The release profiles of DOX@[email protected]

in PBS buffers (pH = 7.4 and pH = 5.5) are shown in Figure 6a. It can be seen

that the DOX@[email protected] displayed very low release cumulative amounts (below 25%) until 48 h in pH = 7.4 PBS buffer. However, the release behavior became more easily and faster when the pH of the PBS buffer was changed to 5.5, and the release cumulative amounts reached nearly to 86%. In this case, the pH sensitive properties of the DOX@[email protected] can be


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Figure 6. In vitro drug release. (a) DOX release from DOX@[email protected] at pH 5.5 and 7.4. (b) NIR irradiation triggered DOX release from DOX@[email protected]. (c) Intracellular DOX release from DOX@[email protected] after incubation for 2 h with and without NIR laser irradiation (1.0 W/cm2, scale bar = 50 μm). The control group is free of DOX@[email protected]. The blue color of DAPI is used to label cell nucleus, while the DOX shows a red color.

attributed to the detachment of the PDA layer from the surface of the TiO2-x in the acidic condition.41 To verify its NIR-triggered release feature, the release profiles of DOX@[email protected]

were studied under different pH conditions (pH = 7.4 and 5.5) with NIR laser

irradiation occasionally. In Figure 6b, both of the two release curves show the “on-off” drug release properties responding to NIR irradiation. The intervention of NIR irradiation accelerated the drug release prominently, especially in pH = 7.4 PBS buffer. The NIR triggered “on-off”


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drug release was attributed to the enhanced photothermal efficiency of [email protected] Moreover, the TEM and dynamic light scattering (DLS) measurement results of DOX@[email protected] were examined after NIR irradiation triggered DOX release test shown in Figure 6B. From TEM images, we can see the PDA layer detachment from the surface of the TiO2-x for both of above two groups. (Figure S6) In addition, the hydrodynamic diameters of DOX@[email protected] decreased from 719.3 nm to 644.7 nm for NIR laser/pH 5.5 and 654.4 nm for NIR laser/pH 7.4, respectively. (Figure S6) Therefore, we concluded that both the pH and photothermal effect could accelerate the detachment of the PDA layer from the surface of the TiO2-x. NIR-triggered DOX release was evaluated further at cellular level by a confocal laser scanning microscopy. The blue color of DAPI was used to label cell nucleus, while the DOX showed a red color. As can be seen from Figure 6c, very weak red fluorescence is seen from in MDA-MB-231 cells after incubation with DOX@[email protected] for 2 h, indicating that the PDA layer has a good sealing effect on DOX. Upon the NIR irradiation, the intracellular red fluorescence enhanced significantly due to the DOX releasing, which may be attributed to thermally driven diffusion process and thermodestruction on PDA layer. We next studied the antitumor mechanism of the TiO2-x based nano-platform under the NIR laser irradiation. As to chemotherapy drug of DOX, the anti-tumor mechanism is intensively reported as via intercalating into DNA and halting transcription.43 The PTT and PDT induced cells apoptosis by TiO2-x matrix may lead to detrimental biochemical changes, such as dysfunction of mitochondria and membrane permeability.44 Firstly, we checked the phototherapeutic disturbance on mitochondria membrane potential (MMP) by the JC-1 staining. JC-1 is a green-fluorescent monomer at low membrane potential, while it forms red-fluorescent JC-1 aggregates at higher potentials. In the apoptotic cells, JC-1 situates at the cytoplasm and


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gives a green fluorescence, while it aggregates within the mitochondria in healthy cells to emit a red fluorescence. Thus, the ratio of red to green fluorescence reflects the degree of MMP changes. As illustrated in Figure 7, the control groups of untreated cells, TiO2-x@PDA treated cells and NIR treated cells show red fluorescence but no green color, suggesting that nearly no MMP changes occurred in these cells. In a sharp contrast, strong green fluorescence emerged from the cells that mediated by both of TiO2-x@PDA and NIR irradiation, and it indicates that hyperthermia and ROS during the phototherapy process damaged cancer cells by mitochondria dysfunction to a certain degree. Secondly, the influences of phototherapy on membrane permeability were studied by ethidium bromide (EB) staining, which is fluorescent molecule and impermeable to normal cells. As membrane permeability changed, it can stain the dead/apoptosis cells by binding to cell nucleus to emit red fluorescence. From the Figure S7, TiO2-x@PDA mediated phototherapy can greatly change the cell membrane permeability and make it even penetrable to EB. However, the control group, TiO2-x@PDA and NIR treated groups are not in this case as no ethidium bromide (EB) fluorescence can be seen. Consequently, the antitumor mechanism of our nano-platform may involve DOX-induced DNA damage and PDT/PTT caused mitochondrial dysfunction/change of cell membrane permeability, as well as synergistic therapeutic effects between them.


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Figure 7. The detection for mitochondrial potential changes of different groups by JC-1 staining. (Scale bar = 20 μm) Above in vitro anti-tumor studies disclosed a significantly enhanced cancer cell inhabitation efficacy by our synergistic treatment system, which encourages us to go a further step of in vivo cancer treatment. We firstly monitored the temperature elevation in tumors under NIR laser irradiation. The in vivo thermographic images and relevant temperature profiles of tumor-bearing mice were achieved by an IR thermal camera. As depicted in Figure 8a-b, the surface temperature of tumor site only increases about 4 oC after 10 min NIR irradiation. However, it could be greatly improved by cooperating with photothermal agents, such as TiO2-x@PDA or DOX@[email protected]. The temperature of NIR mediated TiO2-x@PDA and DOX@[email protected]

groups reached up to 53.5 °C and 58.5 °C, respectively, which could lead to


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tumor cell apoptosis even within a short time exposure of a few minutes. The in vivo therapeutic effects of DOX@[email protected] were then evaluated on tumor bearing mice by measuring the inhibitory rate of tumor. In the Figure 8c-d, the tumor volumes of mice received only TiO2x@PDA

or NIR irradiation treatment show no conspicuous difference to that of control group

without any treatment, all of which present rapid growth of the solid tumors, indicating that neither material itself or NIR irradiation could give rise to antitumor effect. It seems to be effective for the NIR mediated TiO2-x@PDA group, which triggered PDT and PTT effects, at the first 10 days. However, the volume of the tumors increases again after that time, suggesting that the hyperthermia and ROS produced during the phototherapy are not effective enough for completely damaging the whole tumor tissue, especially for inside part of tumor. When the DOX was introduced into the system, the NIR mediated DOX@[email protected] displayed a remarkable tumor inhibition effect and some tumors even vanished after the treatment. The onsite release of DOX during the phototherapy made an additional antitumor effect. In addition, hyperthemia driven DOX diffusion can permeate the whole tumor and remedy the weaknesses of PDT/PTT that has a poor therapeutic effect to tumor in depth.


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Figure 8. In vivo anti-tumor studies. (a) Infrared thermal images of MDA-MB-231 tumorbearing mice and (b) corresponding temperature profiles. (c) Quantitative measurement of tumor volume in mice. (d) Representative photographs of mice and tumors. (e) Body weight changes of mice during the treatment. (f) H&E staining of tumor slices collected from different groups of mice after 14 days treatment.

As shown in Figure 8e, the mice in the five groups show no obvious weight losses, implying that therapeutic method and materials in this work have no acute toxicity towards the mice. After the treatment, the histological sections of tumor and major organs were studied by hematoxylin and eosin (H&E) staining. In the Figure 8f, there is nearly no obvious tumor cell necrosis for


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control, TiO2-x@PDA or NIR treated ones. While for the NIR mediated TiO2-x@PDA and DOX@[email protected] groups, sliced tumors exhibited massive cell necrosis and apoptosis. In addition, no remarkable lesion (e.g. necrosis or inflammatory) were found in major organs after the treatment, highlighting the in vivo biosafety of using DOX@[email protected] for cancer treatment (Figure S8). Finally, we employed B-mode ultrasonography (US) to monitor evolution of tumor after treatment. As shown in the B-mode sonogram, the tumor volume gradually decreases from the first day to the seventh day and eventually completely vanished after 14 days’ treatment (Figure 9), which is consistent with previous in vivo anti-tumor observation. Moreover, the color Doppler flow images (CDFI) of different days show low vascularity, which is accorded with the characteristic of MDA-MB-231 tumor model. In the US-elastogram, the stiffness of tumor tissue is high as evidenced by a blue color at first day post treatment. However, the blue-green-red pattern artifact appeared around the tumor at the third day, suggesting that the tumor began to liquefaction necrosis. At the seventh day, the tumor softened further with the bigger swelling area because of the more serious liquefaction necrosis. After 14 days’ treatment, the tumor disappeared and the stiffness of the previous tumor site (red arrow) was nearly the same to the peripheral tissue, indicating the outstanding tumor ablation ability of DOX@[email protected] triple-therapy system.


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Figure 9. B-mode ultrasonography for monitoring evolution of tumor after treatment. The dashed circles indicate the tumor area and the arrows mark the original tumor site.

CONCLUSION In this work, we established a multifunctional theranostic nano-platform based on mesoporous TiO2-x matrix, which is an excellent NIR-harvesting material to simultaneously produce ROS, hyperthermia and PAT signal under NIR excitation. Moreover, the mesoporous structure of TiO2x

matrix also endowed it to be an adequate drug carrier with controllable capability of drug

release.

As

a

result,

the

composited

DOX@[email protected]

system

realized

fluorescence/PAT dual-mode bio-imaging and triple combination therapy of PDT/PTT/ chemotherapy. Both of in vitro and in vivo anti-tumor study revealed the superiority of triple combination therapy, which can greatly inhibit the tumor growth and even thoroughly ablate the solid tumor. The antitumor mechanism was confirmed as a synergistic action of DOX-induced


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DNA damage and PDT/PTT caused mitochondrial dysfunction/change of membrane. B-mode ultrasonography certified the elimination of solid tumor via a liquefaction necrosis process.

ASSOCIATED CONTENT Supporting Information. The membrane permeability study of MDA-MB-231 cells of different groups and Histology staining of organ slices (liver, kidney, heart, lung and spleen) tumor slices collected from different groups of mice after 14 days’ treatment. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (C. S. Guo), [email protected] (S. Q. Liu) Author Contributions ‡

Wei Guo and Fei Wang contributed equally.

Funding Sources

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (No.51572059). REFERENCES (1)

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TiO2âx Based Nanoplatform for Bimodal Cancer Imaging and NIR

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