Activating TiO2 Nanoparticles: Gallium-68 Serves as a High-Yield

Jan 25, 2018 - The classical photodynamic therapy (PDT) requires external light to activate photosensitizers for cancer treatment. However, limited ti...
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Activating TiO2 Nanoparticle: Gallium-68 Serves As A High-Yielded Photon Emitter for Cerenkov Induced Photodynamic Therapy Dongban Duan, Hui Liu, Yang Xu, Yuxiang Han, Mengxin Xu, Zhengchu Zhang, and Zhibo Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17902 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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Activating TiO2 Nanoparticle: Gallium-68 Serves As A High-Yielded Photon Emitter for Cerenkov Induced Photodynamic Therapy Dongban Duan,† Hui Liu,† Yang Xu,† Yuxiang Han,† Mengxin Xu,† Zhengchu Zhang,† Zhibo Liu*,†,‡ †Beijing National Laboratory for Molecular Sciences, Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China ‡Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China *Corresponding author: Zhibo Liu. E-mail address: [email protected] Keywords: Cerenkov radiation, photodynamic therapy, titanium dioxide, Gallium-68, positron emission tomography ABSTRACT: The classical photodynamic therapy (PDT) requires external light to activate photosensitizers for cancer treatment. However, limited tissue penetration of light has been a long-standing challenge for PDT to cure malignant tumor in deep tissue. In recent, Cerenkov radiation (CR) emitted by radiotracers such as 18

F-fluorodeoxyglucose (18F-FDG) has become an alternative and promising internal

light source. Nevertheless, fluorine-18 (F-18) only releases 1.3 photons per decay in average, consequently injection dose of F-18 goes beyond 10-30 times more than usual to acquire therapeutic efficacy due to its low Cerenkov productivity. Gallium-68 (Ga-68) is a favorable CR source owing to its readily availability from generator and 30-time higher Cerenkov productivity. Herein, we report as the first time of using Ga-68 to be a CR source to activate dextran modified TiO2 nanoparticles (D-TiO2 NPs) for CR induced PDT. Compared with

18

F-FDG, Ga-68 labeled bovine serum

albumin (68Ga-BSA) inhibited the growth of 4T1 cells and exhibited significantly stronger DNA damage to tumor cells. In vivo studies showed that the tumor growth was almost completely inhibited when tumor-bearing mice were treated with a combination of D-TiO2 NPs and 68Ga-BSA. This study proved that Ga-68 was a more potent radionuclide for PDT than F-18 both in vitro and in vivo, and offered a promising strategy of using diagnostic dose of radioactivity to achieve

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depth-independent cancer therapy without using any external light source. INTRODUCTION Photodynamic therapy (PDT), a noninvasive and effective medical technique, has emerged as an attractive cancer treatment both in clinical research and practice.1-3 In general, external light source is needed for PDT to activate photosensitizers (PSs), generating cytotoxic reactive oxygen species (ROS) and destroying targeted cells and tissues.4-6 Treating cancers on skin and other epidermal tissue by external light excited PDT has been widely used in clinics.7 However, external light suffers from the rapid attenuation through tissue, therefore can hardly reach the malignant lesions hiding under deep tissue, makes PDT with nearly impossible.8-11 A recent approach that utilizing Cerenkov radiation (CR) as an internal light source has shed the light on overcoming the limitation of tissue penetration of light.12-14 Cerenkov radiation happens when charged particles, such as β+ and β-, travel through a dielectric medium beyond the speed of light. The emitting spectrum is continuous, includes ultraviolet (UV) and visible light, and can be defined by Frank–Tamm formula.15 The dominant emission of CR is UV which is ideal for UV responsive photosensitizer such as titanium dioxide (TiO2)12 or chlorin e6,16 both of which have been widely used in PDT. These findings are revolutionary because radionuclides can be specifically delivered to desired lesion in the body by target-specific radiotracers,17-18 meaning CR can be an internal light source that immediately stimulates photosensitizer in situ without passing through any tissue.19 It is commonly accepted that the success of PDT depends on the intracellular concentration of ROS, which is determined by local photon intensity as ROS is the product from the interaction between photon and PS.20-21 Consequently, high intensity of CR is favored in order to provide sufficient ROS and thereafter cause cell damage. The CR productivity varies significantly from different radionuclides. For example, in aqueous solution, Y-90 decays to release an electron with the average energy of 2,280 keV and gives 47.3 photons per decay; Ga-68 decays to release a positron with the average energy of 1,899 keV and gives 33.9 photons per decay; F-18 decays to release

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a positron with the average energy of 633 keV and gives 1.32 photons per decay; Cu-64 decays to release an electron with the average energy of 574 keV and gives 0.56 photons per decay,22 demonstrating their productivity of CR and the related ROS can amplify magnitudely when β-emitters with higher energy are employed. Radionuclides such as Cu-64, F-18 and Ga-68 have been widely used in clinics for positron emission tomography (PET).23-28 Among them, Ga-68 has about 20 times higher CR productivity than the others. In addition, Ga-68 labeled PET tracers such as 68

Ga-PSMA,29

68

Ga-DOTA-TATE30 and

68

Ga-DOTA-TOC31 have been extensively

used in cancer diagnose, and exhibit high specificity on tumor targeting. Furthermore, Ga-68 is readily available from

68

Ge/68Ga generator without the need of an on-site

cyclotron.32-33 These three traits synergize to an optimal radionuclide for CR induced PDT. Herein, we report as the first time of using Ga-68 as a CR source to activate TiO2 NPs for depth-independent PDT. 18F-FDG was also used as a low CR yield tracer for comparison. In order to coordinate with the ultraviolet-weighted spectrum of CR, titanium dioxide (TiO2) was applied as an UV responsive photocatalyst. Upon irradiation with UV light, electron–hole pairs are generated in TiO2 and further react with water or oxygen to form ROS.34-37 As an internal UV light source, CR emitted by 18

F-FDG or 68Ga-BSA activate nearby TiO2 NPs to generate cytotoxic ROS. Notable

suppression on cell growth of 4T1 cells was observed in vitro with 68Ga-BSA. Animal studies were carried out in a murine breast tumor model by subcutaneous injection of D-TiO2 NPs and PET tracers. Comparing with 18F-FDG, 68Ga-BSA was significantly more effective on cancer treatment in vivo and the volume of xenografts was under control. In general, a long half-life radionuclide is preferred for cancer treatment, but this work reports a novel application of Ga-68, of which the half-life is only 68 minutes, to treat cancer successfully via CR induced PDT. A head-to-head comparison between Ga-68 and F-18 on cancer treatment was described in details, indicating the importance of employing the radionuclides with higher CR yield rather than longer half-life. In conclusion, Ga-68 was unveiled as a suitable radionuclide for CR induced PDT after detailed experimental assessment in cooperation with a TiO2 nano-photosensitizer.

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EXPERIMENTAL SECTION Materials. All the chemical reagents were used as received without further purification. Titaniumbutoxide (Ti(OBu)4) and isopropanol were purchased from Sinopharm Chemical Reagent limited corporation (China). Tetraalkylammonium hydroxide (TMAOH), carboxymethyl dextran sodium salt (CMD) (Mw = 10 kDa), dopamine·HCl, fluorescin isothiocyanate (FITC), and 1,3-diphenylisobenzofuran (DPBF)

were

purchased

from

Aladdin

(China).

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide·hydrochloride

(EDC),

N-hydroxysuccinimide (NHS) and bovine serum albumin (BSA) were purchased from J&k (China). Mental free hydrochloric acid and sodium tartrate were purchased from Sigma-Aldrich. 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was purchased from Macrocyclics, Inc. Cell Counting Kit-8 (CCK-8) was purchased from Biyuntian Biotechnology Institute.

18

F-FDG (3.7

MBq/µL) was provided by Beijing Cancer Hospital (Beijing, China). Mouse monoclonal anti-phosphohistone γ-H2AX was purchased from eBioscience. Synthesis of TiO2 Nanoparticles. TiO2 nanoparticles were prepared via a hydrothermal method according to a literature procedure.38 Briefly, a mixture of Ti(OBu)4 (1.7018 g, 5 mmol) and 8.3 mL of isopropanol was stirred at room temperature under N2 protection for 10 min. The mixture was slowly added into distilled water (13.6 mL) and stirred for 30 min. The obtained precipitate was centrifuged and washed with excess distilled water three times. The precipitate was resuspended in 50 mL of 0.1 M TMAOH aqueous solution and maintained at 70 ℃ for 1 h. The as-formed reaction solution was transferred into a 100 mL sealed Teflon autoclave and maintained at 240 ℃ for 2 h. After cooling the autoclave to room temperature, the obtained precipitates were separated by centrifugation and washed with deionized water two times. The as-synthesized TMAOH-TiO2 NPs were dispersed in bacteria-free water and stored at 4 ℃. Surface Modification of TiO2 Nanoparticles. First, dopamine conjugated CMD

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(D-CMD) was prepared via EDC-NHS chemistry.39 In brief, 400 mg of CMD was dissolved in 10 mL of water, to which 76.7 mg of EDC and 57.5 mg of NHS was added. Subsequently, 1 mL of 0.1M dopamine·HCl aqueous solution was added dropwise and stirred over night at RT. The obtained solution was dialysed against water using 3 kDa cut-off membrane. The dialysate was freeze-dried and stored at 4 ℃. Second, 2 mL of TiO2 NPs aqueous solution (5 mg/mL) was added dropwise into 50 mg of D-CMD (10 mg/mL) aqueous solution. After vigorous stirring for 2 h, DA-CMD-modified TiO2 NCs was separated by centrifugation and washed with deionized water several times to remove the unreacted D-CMD. Finally, the samples were suspended in 1 ml bacteria-free water and stored at 4 ℃. Characterization. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) experiments were performed on a JEM-2100F microscope (JEOL Ltd., Japan). Nuclear magnetic resonance (NMR) spectra was recorded on Bruker Avance Spectrometers (400 MHz). The hydrodynamic size and zeta potential was characterized by Nanobrook Omni (New York, U.S.A.). Powder X-ray (XRD) patterns were recorded on a SIEMENS D 5005 X-ray diffractometer. UV-vis spectrum measurements were recorded on a NanoDrop one (ThermoFisher, U.S.A.). Flow cytometry analysis was performed on a BD LSR Fortessa (BD Bioscience. U.S.A.). Fluorescence images were taken on a Nikon A1R confocal laser scanning microscope. UV-Driven Photocatalytic Experiment. In a typical photocatalytic test, 5 mg D-TiO2 NPs was mixed with 2 mL of DPBF solution and kept in the dark for 12 h before irradiation. Then the mixture is exposed to WD-9403E (365 nm ultraviolet lamp, 5 mW/cm2, China) with magnetic stirring. After irradiation for different times, D-TiO2 NPs were separated by centrifugation. The concentration of DPBF was determined by UV-vis absorption spectrum at 420 nm. To confirm the in vitro PDT effect, 4T1 cells incubated with D-TiO2 NPs were irradiated with ultraviolet lamp (5 mW/cm2) for 30 min. The cell viability assay was conducted after 12h incubation. Conjugation

of

p-SCN-Bn-NOTA

to

BSA.

10-fold

molar

excess

of

p-SCN-Bn-NOTA chelator was added to BSA in PBS buffer (pH=8) and incubated

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for 1 h at room temperature. The obtained product of BSA-NOTA was dialysed against water and stored at -20 ℃. Synthesis of 68Ga-BSA. 68Ga was eluted from a 68Ge/68Ga generator using 0.6 M HCl. The peak fraction (1.0 mL, 1.1-1.5 GBq) was mixed with NaOH (190 µL, 3 M) and sodium acetate solution (200 µL, 1 M) to adjust the pH value to 5.5. Then the mixture was transferred to a 1.5 mL Eppendorf tube containing 1 mg of BSA-NOTA and incubated for 5 min at 37 ℃.40 Subsequently, the product was purified by gel filtration on a disposable PD10 column, equilibrated with saline. Cell culture. Mouse breast cancer 4T1 cells were maintained in RPMI 1640 (Corning, Manassas, U.S.A.) supplemented with 10% FBS, 100 U mL-1 of penicillin and 100 U mL-1 of streptomycin. The cells were incubated in a humidified incubator with 5% CO2 at 37 ℃. Cell uptake studies of 18F-FDG and 68Ga-BSA. 4T1 cells (1×105 per well, triplicate for each group) were plated in 24-well plates and incubated at 37 °C overnight. The cells were then incubated for various times (0.5, 1, 1.5, 2.0, 2.5 and 3 h) at 37 °C with 0.37MBq

18

F-FDG or

68

Ga-BSA in serum-free medium, respectively. At designated

time points, radioactive medium was collected and cells were lysed with 0.1 M NaOH for 5 min at room temperature. The radioactivity of the medium and cell lysates was counted by a γ-counter (FH463B, China National Nuclear Corporation). Cell-viability assays. Cell viability was assessed with the CCK8 proliferation assay following the manufacturers’ protocol. Treatment groups were normalized to controls. Each assay was repeated 3 times. In Vitro CR-Induced PDT. Cells were seeded in a 96-well plate at a density of 5000 cells per well 12 h prior treatment. The cells were incubated with D-TiO2 NPs (100 µg/mL) for 12 h. After incubation with D-TiO2 NPs,

18

F-FDG or

68

Ga-BSA was

added at 100, 200, 400 µCi/well. After 48 h incubation, cells were washed with PBS and the cell viability was analyzed. γ-H2AX Immunofluorescence Analysis. 4T1 cells seeded in 12-well plates were incubated with D-TiO2 NPs (100 µg/mL) for 12h. Then 400µCi of 68

18

F-FDG or

Ga-BSA was added and incubated for another 12h. After incubation, the cells were

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fixed by 4% of paraformaldehyde for 10 min and washed with PBS. The γ-H2AX immunofluorescence staining assay was then conducted following the standard protocol.41 Confocal fluorescence images were visualized with confocal laser scanning microscope. Tumor Model. Six-week old female BALB/c mice were ordered from Vital River Laboratories (Beijing, China) and kept under Specific Pathogen Free (SPF) condition with free access to standard food and water. Approximately 2×106 Murine breast cancer 4T1 cells suspended in 100 µL of PBS were implanted subcutaneously into the right shoulder of BALB/c mice. Tumor volumes were calculated with the formula: the volume = length× width2/2. All animal experiments were performed in accordance with guidelines approved by the ethics committee of Peking University. In Vivo CR induced PDT Cancer Treatment. When the tumor volumes were approximately 50 mm3 (∼7 days after tumor inoculation), the first treatment was conducted, and the same treatment was conducted two days later (∼9 days after tumor inoculation). The treatment protocols were described as follows. D-TiO2 NPs (20 mg/mL, 50 µL) were injected directly into the tumor mass under anesthesia, and 30 MBq of 18F-FDG or 68Ga-BSA was injected into the tumor mass 12 hours later. The mice were monitored for 30 days after treatment. The tumor volumes of mice were measured with callipers every two days, and the weight of mice was also monitored. For survival analysis, mice were euthanized by cervical dislocation after anaesthesia with 5% isoflurane when the tumor size reached 1000 mm3. Small-Animal PET/CT Imaging. 4T1 tumor bearing mice were intratumoral injected with

18

FDG or

68

Ga-BSA (30 MBq, 50 µL). PET imaging was recorded on a

micro-PET/CT scanner (Mediso Medical Imaging Systems). Three hours PET acquisition was acquired for dynamic reconstruction. Three-dimensional regions of interest (ROIs) were drawn on the tumor site to obtain the uptake dose and radioactivity of the tracer. Histology. For the histopathologic study, the major organs including heart, liver, spleen, lung, and kidney were excised from mice 14 days post injection. Organs were fixed in 4.0% paraformaldehyde, embedded with paraffin, sectioned into slices, and

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stained with hematoxylin and eosin (H&E). Samples were chosen at random and the images were acquired by bright-field microscopy. Statistical Analysis. Statistical analyses were performed using GraphPad Prism 6 and Origin 8. Statistical comparisons were analyzed using two-way ANOVA. *p