Lanthanide-Doped CoreâShellâShell Nanocomposite for Dual

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Lanthanide-doped core-shell-shell nanocomposite for dual photodynamic therapy and luminescence imaging by a single X-ray excitation source Chang-Chieh Hsu, Syue-Liang Lin, and Cheng Allen Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00015 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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ACS Applied Materials & Interfaces

Submitted to: ACS Applied Materials & Interfaces

Lanthanide-doped core-shell-shell nanocomposite for dual photodynamic therapy and luminescence imaging by a single X-ray excitation source Chang-Chieh Hsu† , Syue-Liang Lin‡ , and C. Allen Chang†‡§* ∥

∥

†

Department of Biomedical Imaging and Radiological Sciences, ‡ Department of Biotechnology and Laboratory Science in Medicine, and § Molecular Imaging Research Center (MIRC), National Yang-Ming University, Taipei 112, Taiwan. ∥

These authors contribute equally to this work.

*

Corresponding author. E-mail address: [email protected] (C. Allen Chang).

KEYWORDS: Lanthanide-doped nanoparticles, photodynamic therapy, X-ray, luminescence imaging, theranostics ABSTRACT: Photodynamic therapy (PDT) could be highly selective, non-invasive, and with low side effects as an adjuvant therapy for cancer treatment. Because the excitation sources such as UV and visible lights for most of the photosensitizers do not penetrate deeply enough into biological tissues, PDT is useful only when the lesions locate within 10 mm below the skin. In addition, there is no prior example of theranostics capable of both PDT and imaging with a single deep-penetrating X-ray excitation source. Here we report a new theranostic scintillator nanoparticle (ScNP) composite in a core-shell-shell arrangement, i.e. NaLuF4:Gd(35%),Eu(15%) @NaLuF4:Gd(40%)@NaLuF4:Gd(35%),Tb(15%), which is capable of being excited by a single X-ray radiation source to allow potentially deep tissue PDT and optical imaging with low dark cytotoxicity and effective photocytotoxicity. With the X-ray excitation, the ScNPs can emit visible light at 543 nm (from Tb3+) to stimulate the loaded rose bengal (RB) photosensitizer and cause efficient MDA-MB-231 and MCF-7 cancer cell death. The ScNPs can also emit light at 614 nm and 695 nm (from Eu3+) for luminescence imaging. The core-shell-shell ScNPs are unique for the middle shell to separate the Eu3+ in the core and the Tb3+ in the outer shell to prevent resonance quenching between them and to result in good PDT efficiency. Also, it was demonstrated that although the addition of a mesoporous SiO2 layer resulted in 82.7% fluorescence resonance energy transfer (FRET) between Tb3+ and RB, the subsequent conversion of the energy from RB to generate 1O2 was hampered, regardless the loaded amount of the RB was almost twice that without the mSiO2 layer. A unique method to compare the wt% and mole% compositions calculated by using the morphological TEM images and the inductively coupled plasma (ICP) elemental analysis data of the core, core-shell and core-shell-shell ScNPs is also introduced.

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1. INTRODUCTION Surgery, chemotherapy and radiation therapy are the most common traditional ways to treat cancer.1 However, because these treatments are either invasive or with many side effects, alternative therapies are currently under clinical development. Among them, the less-invasive photodynamic therapy (PDT) which employs light-excited photosensitizers to convert triplet oxygen molecules (3O2) into singlet oxygen molecules (1O2) or to produce other reactive oxygen species (ROS) to kill cancer cells, could be more selective and safer.2,3 However, PDT is mostly useful for skin and shallow-tissue treatment due to the limited penetration depth of the excitation light.4 Thus, many solutions have been sought to improve the light penetration depth for PDT,5 including the use of gold nanorods with two-photon laser radiation6 and up-conversion lanthanide nanoparticles with near IR light radiation,7,8 and so far the best penetration depth achieved is 12 mm under the skin.4 Because X-ray has excellent tissue penetration depth, the use of X-ray as the excitation source for PDT to overcome the penetration problem has been proposed, in addition to its computed tomographic (CT) imaging functions.4,9-13 In addition, because the efficiency of 1O2 generation by organic photosensitizers excited by X-ray is normally poor, the use of scintillator nanoparticles (ScNPs) to convert X-ray into visible or near-infrared light suitable to stimulate the photosensitizers is also proposed to overcome the efficiency problem.11 Combining radiation therapy and X-ray induced PDT to treat cancer by employing Tb3+containing ScNPs and porphyrins was firstly investigated by Chen et al. in 2006.10 The use of lanthanide-doped oxide nanoparticles and meso-tetra(4-carboxyphenyl) porphine (MTCP),11 as well as other photosensitizers such as zinc oxide14 and SrAl2O4:Eu2+ together with MC54015 for PDT have also been reported. In addition, X-ray induced optical/luminescence imaging study using NaGdF4:Eu3+ ScNPs was later reported in 2014.16 Although some initial research improvements on the PDT penetration depth and 1O2 generation efficiency have been made using X-ray as the excitation source and ScNPs as light mediators, there are still a number of problems to be solved including: (1) lack of quantitative analysis on the loading of photosensitizers and related quantum efficiency measurement data for future dosage design;17 (2) less efficient 1O2 generation using some of the photosensitizers such as MTCP as well as poor fluorescence resonance energy transfer (FRET) from the ScNPs to the photosensitizers;11,18 and (3) the demand for the development of theranostics combining the diagnosis and treatment in the same time using a single X-ray excitation source. Note that several theranostic agents for PDT and luminescence imaging have been reported using a single near infrared (NIR) excitation, e.g. 660 nm or 980 nm, the problems of less efficiency, shallow 19,20 penetration depth and overheating would still need to be overcome. In this study, we report the synthesis and characterizations of a novel core-shell-shell structured ScNPs NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%)@NaLuF4:Gd(35%),Tb(15%) (core-shell-shell ScNPs) which could be excited by X-ray and simultaneously generates 614 nm and 695 nm red light (by Eu3+) for luminescence imaging as well as 543 nm green light (by Tb3+) and through FRET to excite the loaded photosensitizers rose bengal (RB) to produce 1O2 for PDT.21 Note that RB is a water-soluble photosensitizer with a high absorption coefficient (~


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100,000 M−1cm−1 at 549 nm) and an efficient 1O2 quantum yield as high as 0.75.21,22 It is already used as a topical diagnostic agent for human dry eye symdrome and therapeutic agent in clinical trials for melanoma and oral squamous cell carcinoma with little side effects.23,24 Most importantly, the main absorption band of RB at 549 nm overlaps almost exactly with the main emission band of our core-shell-shell ScNPs at 543 nm which would allow efficient FRET from the core-shell-shell ScNP donor to the RB receptor. 2. EXPERIMENTAL SECTION 2.1. Materials. All the chemical reagents are of analytical grade and used without further purification, including cetyltrimethylammonium chloride (CTAC, T.C.I, Japan), europium(III) acetate hydrate, gadolinium(III) acetate hydrate, terbium(III) chloride hexahydrate, poly(allylamine) (PAH), sodium hydroxide, ammonium fluoride, 1-octadecene, oleic acid, tetraethyl orthosilicate (TEOS), 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA, Sigma-Aldrich, USA), lutetium(III) acetate hydrate (Alfa Aesar, USA), phosphate buffered saline (PBS, Omic Biosystems, Taiwan), hoechst 33342 (nuclear counterstain for fluorescence microscopy, Enzo Life Sciences,USA), rose bengal (RB), cell counting Kit 8 (Sigma-Aldrich, USA), folic acid-PEG3k-NHS (NANOCS, USA), Dulbecco's Modified Eagle Medium (DMEM, Corning, USA). Double-distilled water was used for all related experiments. 2.2. Synthesis of the core ScNPs NaLuF4:Gd,Eu. The synthesis of the core ScNPs doped with 5%, 10% and 15% Eu3+ was accomplished by a thermal decomposition method.25 In a typical synthesis of NaLu(50%)F4:Gd(35%),Eu(15%), appropriate amounts of Gd(CH3CO2)3·4H2O (0.142 g, 0.35 mmole), Eu(CH3CO2)3·4H2O (0.0629 g, 0.15 mmole) and Lu(CH3CO2)3·4H2O (0.212 g, 0.5 mmole) with 35%, 15%, and 50% molar ratios were dissolved in 10 mL oleic acid and 15 mL 1-octadecene. The solution was stirred at 190 °C for 1 h under an argon atmosphere, cooled to 65 °C, and NH4F (0.15 g, 4.0 mmole) and NaOH (0.1 g, 2.5 mmole) were added and the solution was stirred for another 1 h. Finally, the solution was heated to 300 °C for 1 h, and the NaLuF4:Gd(35%),Eu(15%) product was obtained by centrifugation, and was kept in 15 mL n-hexane. Yield 85 %. Elemental analysis data (ICP, ppm): Lu, 21.50; Gd, 14.60; Eu, 6.11. 2.3. Synthesis of the core-shell ScNPs NaLuF4:Gd,Eu@NaLuF4:Gd. The first shell NaLuF4:Gd(40%) was grown over the core NaLuF4:Gd(35%),Eu(15%) ScNPs using a seedmediated method.16 Lu(CH3CO2)3·4H2O (0.127 g, 0.3 mmole) and Gd(CH3CO2)3·4H2O (0.0812 g, 0.2 mmole) with 60% and 40% molar ratios were dissolved in 10 mL oleic acid and 15 mL 1octadecene. The solution was stirred at 190 °C for 1 h under an argon atmsophere, cooled to 65 °C, and NH4F (0.075 g, 2.0 mmole) and NaOH (0.05 g, 1.25 mmole) were added and the solution was stirred for another 30 min. Then, 7.5 mL pre-prepared core NaLu(50%)F4:Gd(35%), Eu(15%), dispersed in n-hexane, was added and the mixture was stirred for another 30 min before heating to 300 °C for 1 h. After cooling to room temperature, the core-shell product NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%) was obtained by centrifugation, and was kept in 15 mL n-hexane. Yield 75%. Elemental analysis data (ICP, ppm): Lu, 28.90; Gd, 18.90; Eu, 4.26. 2.4. Synthesis of the core-shell-shell ScNPs NaLuF4:Gd,Eu@NaLuF4:Gd@NaLuF4:Gd, Tb. The second shell NaLuF4:Gd(35%),Tb(15%) was grown over the core-shell nanoparticles


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using a similar seed-mediated method. Lu(CH3CO2)3·4H2O (0.106 g, 0.25 mmole), Tb(Cl)3·6H2O (0.028 g, 0.075 mmole), and Gd(CH3CO2)3·4H2O (0.07105 g, 0.175 mmole) with 50%, 15% and 35% molar ratios were dissolved in 10 mL oleic acid and 15 mL 1-octadecene. The solution was treated in the same way as that for the core-shell synthesis. The final coreshell-shell product NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%)@NaLuF4:Gd(35%),Tb(15%) nanoparticles (ScNP) was obtained by centrifugation, and was kept in 15 mL n-hexane. Yield 71%. Elemental analysis data (ICP, ppm): Lu, 28.70; Gd, 19.39; Eu, 2.21; Tb, 2.75. 2.5. Surface modification of ScNPs with mSiO2 and poly(allylamine) (PAH).26-28 Cetyltrimethylammonium chloride (CTAC, 0.5 g) and the core-shell-shell ScNP (2.5 mg) were dissolved in 20 mL water at 50 °C under stirring overnight. The mixture was sonicated for 30 min and 0.1 mL tetraethyl orthosilicate (TEOS), 100 µL 0.1 M NaOH and 1.5 mL ethyl acetate were added dropwise, and the resulting mixture was stirred for 5 h at 70 °C. The rude product was collected by centrifugation and washed twice with 5 mL ethanol to remove the residual reactants. To remove the CTAC, the rude product was further extracted for 2 h with ammonium nitrate (NH4NO3, 0.3 g) in 50 mL ethanol at 60 °C for 2 h, and cooled to room temperature. The ScNP-mSiO2 product was then obtained by centrifugation and washed with water 3 times. To obtain amino-functionalized ScNP or ScNP-mSiO2, the ScNP or ScNP-mSiO2 (5 mg) was added to a mixture of 10 µL (20 wt%) of PAH diluted with 10 mL of ethanol and the mixture was stirred for 24 hours at room temperature. After centrifugation, the ScNP-PAH or ScNP-mSiO2PAH product was collected and re-dispersed in water for further use. 2.6. Loading and quantitation of the photosensitizer rose bengal (RB) on ScNPs.15 An electrostatic interaction approach was adopted to load RB onto ScNPs by mixing 10 mg ScNPPAH or 10 mg ScNP-mSiO2-PAH with 2 mg RB in water and the mixture was stirred for 1 h and then centrifuged to obtain the final adducts. Quantitation of the loaded RB was performed by spectrometry to be 9.63 ± 0.18 and 19.02 ± 0.09 (wt%) for ScNP-PAH-RB and ScNP-mSiO2PAH-RB, respectively, using a pre-prepared calibration curve (Figure S1, Supporting information). 2.7. Covalent conjugation of ScNP-PAH-RB and ScNP-mSiO2-RB with NHS-PEG3kfolic acid.28,29 In order to improve the biocompatibility and potential targeted ability, NHSPEG3k-folic acid (FA) was conjugated covalently to the core-shell-shell ScNP-PAH-RB and ScNP-mSiO2-PAH-RB. In a typical experiment, 10 mg of NHS-PEG3k-folic acid and 10 mg of ScNP-PAH-RB or ScNP-mSiO2-PAH-RB were added into 10 mL of water. The mixture was stirred for 24 h at room tempurture, then the solution was centrifuged to obtain ScNP-PAH-RBPEG-FA or ScNP-mSiO2-RB-PEG-FA. The products were then washed with cold water and dispersed in water for further use. 2.8. Singlet oxygen determination under X-ray irradiation. 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) was used to determine the generated singlet oxygen after X-ray radiation on the RB modified ScNPs according to a published method.30 In a typical experiment, 10 µM of a ABDA–DMSO solution was added to 2 mL of ScNP-PAH-RB-PEG-FA or ScNP-mSiO2-RB-PEG-FA aqueous solution. The mixture was then irradiated using a RS2000 X-ray irradiator at 2, 4, 6, 8, and 10 Gy, and the fluorescence intensities of ABDA at 407 nm were measured in triplicates to correlate with the amounts of singlet oxygen generated.


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Values are means ± SD (n=3). 2.9. Cytotoxicity assays.31,32 The cytotoxity of the core-shell-shell ScNP-PAH-RB-PEG-FA was evaluated using MDA-MB-231 and MCF-7 cancer cells. The cells were seeded in 96-well plates (1 × 104 cells per well). After incubation for 24 h at 37 °C under 5% CO2, 100 µL of ScNP-PAH-RB-PEG-FA with different concentrations (i.e. 0, 5, 10, 20, 25, 50, 100, 200, and 400 µg/mL) were added into each well and incubated for 24 h. Then, 50 µl CCK-8 reagent (10 times dilution with DMEM) was added into each well. After incubation for 1.5 h at 37 °C, the cell viability was determined by measuring the absorbance at 450 nm in each well. Values are in mean ± SD (n=5). 2.10. In vitro X-ray induced PDT. MDA-MB-231 and MCF-7 cancer cells were seeded in 96-well plates (1 × 104 cells per well) and incubated for 24 h at 37 °C under 5% CO2. Then the core-shell-shell ScNP-PAH-RB-PEG-FA (50 µg/mL) was added into each well and incubated for another 24 h. The cells (with ScNPs) and the control group cells (without ScNPs) were then placed into a RS-2000 biological irradiator under 160kV at 25 mA, and irradiated with 1, 3, 5 Gy X-ray, followed by incubation for another 24 h and addition of the CCK-8 reagent (50 µl, 10 times dilution with DMEM) to each well. After incubation for 1.5 h at 37 °C, the cell viability was determined by measuring the absorbance at 450 nm in each well. Values are in mean ± SD (n=4). 2.11. Photosensitizer release test.15 The photosensitizer release test in different pH was performed in 0.01 M 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5), PBS (pH 7.4) and carbonate-bicarbonate (pH 8.6) buffer solutions for the core-shell-shell ScNP-PAH-RB-PEG-FA. The absorption sepctra of ScNP-PAH-RB-PEG-FA (10 µg/mL) were measured in the UV-Vis region before and after storing in different pH buffer solutions for 24 h. Each solution was then centrifuged and the ScNP-PAH-RB-PEG-FA was re-dispersed in fresh buffer solution and the absorption sepctrum was measured again. The differences in absorbance at 549 nm were used to estimated the released RB percentages. 2.12. In vitro cancer cell imaging. MDA-MB-231 and MCF-7 cancer cells were seeded into six-well culture dishes at a concentration of 5 × 105 cells per well (2 mL) and incubated for 24 hours at 37 °C under 5% CO2 with and without the addition of the core-shell-shell ScNP-PAHRB-PEG-FA (300 µg). All the cells were then incubated for 4 h and washed with the PBS buffer solution to fully remove any excess ScNP-PAH-RB-PEG-FA. The cells were fixed by adding para-formaldehyde (2 wt%, 1 mL) in each culture dish for 10 minutes and the cell nuclei were stained with hoechst 33342 for 10 minutes. After washing with PBS solution, the cells were imaged using a laser confocal microscope (Olympus FV1000 & ZEISS LSM 880). 2.13. Physical and spectral characterizations. Powder X-ray diffraction (XRD) measurements were performed on a Bruker D8 X-ray Powder Diffractometer at a scanning rate of 15°/min in the 2θ range of 10° to 80°. The morphological images of ScNPs were recorded on a transmission electron microscope (TEM, JEM-2000EXII) and a high-resolution transmission electron microscope (HRTEM, JEM-2100). Emission spectra in the UV-Visible and near IR ranges were measured using an Edinburgh FSP920 instrument. X-ray luminescence was measured using a Petrick 0.080 MSFF24 X-ray tube as the irradiation source. X-ray induced


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optical imaging was measured by a home-made fluorescence-computed tomography (FT-CT) instrument equipped with an X-ray tube (NanoRay NS082505) and an EMCCD camera (Princeton Instruments ProEM 512B eXcelon). The X-ray induced phototdynamic therapy was performed by using a RS-2000 X-ray irradiator. The RB loading capacity measurements were performed by a UV-8453 spectrophotometer (Agilent 8453). Inductively coupled plasma measurements were carried out on a Agilent 725 ICP-OES apparatus. Dynamic lighting scattering (DLS) measurements were performed on a XS-100 instrument. 3. RESULTS AND DISCUSSION 3.1. Design, synthesis and morphology of nanoparticles. Figure 1 shows the synthetic steps of the core-shell-shell ScNPs. Two shell layers were seeded on the core, providing different functions for medical applications, i.e. the Eu3+ doped in the core can emit the 614 nm and 695 nm red light after X-ray irradiation for optical imaging and the Tb3+ in the outer shell layer can emit the 543 nm green light to trigger photosensitizers for PDT. The middle shell layer was added to prevent the energy back trasfer between Eu3+ in the core and Tb3+ in the outer shell layer.20 The reason for the doped Gd3+ ions in the core, middle-shell and outer-shell was to use them as antenna to transfer the absorbed X-ray induced UV light at 313 nm (6P7/2 → 8S7/2) to Eu3+ and Tb3+ ions.33,34 The use of the NaLuF4 as the base material was to help prepare monodispersive nanoparticles when doped with other Gd3+, Eu3+ and Tb3+ ions and to prevent concentration quenching.35

Figure 1. The design and synthesis of the core NaLuF4:Gd(35%),Eu(15%), core-shell NaLuF4:Gd(35%), Eu(15%)@NaLuF4:Gd(40%), and core-shell-shell NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%)@NaLuF4:Gd(35%),Tb(15%) ScNPs, and with PAH-RB coating with and without a layer of mSiO2. CSS = core-shell-shell ScNPs. In order to optimize the Eu3+ fluorescence emission efficiency in the core for optical imaging, three different Eu3+ molar percentages, i.e. 5%, 10%, and 15%, were used to synthesize the core ScNPs by a thermal decomposition method and characterized. Figure 2(a, b and c) shows the respective transmission electron microscopy (TEM) images of the hexagonal nanoparticles NaLu(60%)F4:Gd(35%),Eu(5%), NaLu(55%)F4:Gd(35%),Eu(10%) and


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NaLu(50%)F4:Gd(35%),Eu(15%) and their mean sizes were estimated to be 27.5 nm, 20.9 nm and 16.9 nm, respectively. This trend of decreasing size with increasing Eu3+ doped percentage is in good agreement with that of NaYF4:Gd,Yb,Er of a previous study.35 Figure 2(d) shows that the emitted luminescence intensities increase with increasing Eu3+ doped percentages at λex = 395 nm, which is also in agreement with that of NaGdF4:Eu of the other previous study.16 Further increase in the Eu3+ doped percentages led to concentration quenching and reduced emitted luminescence intensities.16 More detailed optimization studies could be done at a later stage, if other properties of the material were appropriate for translational development.

Figure 2. TEM images of the core ScNPs: (a) NaLu(60%)F4:Gd(35%),Eu(5%), (b) NaLu(55%)F4:Gd(35%),Eu(10%) and (c) NaLu(50%)F4:Gd(35%),Eu(15%). (d) Emission spectra of NaLu(60%)F4:Gd(35%),Eu(5%), NaLu(55%)F4:Gd(35%),Eu(10%) and NaLu(50%)F4:Gd(35%),Eu(15%) dispersed in n-hexane (λex=395 nm, 2.5 mg/mL each). After finding out the optimal doping percentage of Eu3+ (i.e. 15%) for the core, the coreshell and core-shell-shell ScNPs were prepared by the same thermal decomposition method in two steps. In the first step, the core precursors and the shell components NaGdF4 and NaLuF4 in appropriate molar ratios were used to obtain the core-shell material. In the second step, the coreshell-shell material was prepared by using the core-shell material with the second shell components in appropriate molar ratios. Figure 3(a, b and c) shows that the TEM images of the core, core-shell and core-shell-shell ScNPs were uniform in size with regular hexagonal shapes. The respective sizes of these nanoparticles were estimated to be 16.9 nm (core), 20.5 nm (coreshell) and 24.6 nm (core-shell-shell) from TEM measurements and the thickness of each shell


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was about 1.8-2.0 nm. A separate dynamic laser scattering (DLS) analysis showed that these ScNPs were quite monodispersive with estimated diameters of 21.0 nm, 25.6 nm and 28.9 nm for the core, core-shell, and core-shell-shell materials, respectively, and a very low polydispersity index (PdI) value of 0.121 (Figure 3, e-g). The fact that DLS analyses of particle sizes are larger than those of TEM measurements has been reported previously.36

Figure 3. (a-d): TEM images of (a) core NaLuF4:Gd(35%),Eu(15%), (b) core-shell NaLuF4: Gd(35%),Eu(15%)@NaLuF4:Gd(40%) and (c) core-shell-shell NaLuF4:Gd(35%),Eu(15%)@ NaLuF4:Gd(40%)@NaLuF4:Gd(35%),Tb(15%). (d) HRTEM image of NaLuF4:Gd(35%), Eu(15%)@NaLuF4:Gd(40%)@NaLuF4:Gd(35%,Tb(15%)@mSiO2. (e-h): DLS analysis results of (e) core NaLuF4:Gd(35%),Eu(15%), (f) core-shell NaLuF4:Gd(35%),Eu(15%) @NaLuF4: Gd(40%), (g) core-shell-shell NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%)@NaLuF4: Gd(35%),Tb(15%) and (h) core-shell-shell NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%)@ NaLuF4:Gd(35%),Tb(15%)@mSiO2. A mesoporous silica (mSiO2) shell was further added on the core-shell-shell ScNPs (cf. Figure 1, vide supra). Figure 3(d) shows that the thickness of the mSiO2 shell is about 25 nm in the HRTEM image. In the mSiO2 layer, there are additional pores which could allow more loading of the RB photosensitizers. This material will be compared with that without mSiO2 modification for the loading of RB after coating with PAH, and their abilities to generate 1O2 molecules (vide infra). Figure 4 shows the XRD patterns of the core NaLuF4:Gd(35%),Eu(15%), core-shell NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%) and core-shell-shell NaLuF4:Gd(35%),Eu(15%) @NaLuF4:Gd(40%)@NaLuF4:Gd(35%),Tb(15%). All of them exhibit hexagonal phase but not cubic phase according to the standard pattern of β-NaYF4 (JCPDS. No. 16-0334).37 According to the Scherrer equation,38 τ = Kλ/βcosθ (where τ is the mean size of the ordered (crystalline) domains, K is a dimensionless shape factor with a value close to 0.9-1.0 and varies with the actual shape of the crystallite, λ is the X-ray wavelength, β or ∆(2θ) is the line broadening at full width at half maximum (FWHM), after subtracting the instrumental line broadening in radians, and θ is the Bragg angle), the nanoparticle diameter is inversely proportional to the FWHM value. At 2θ = 17.0, 30.0, 30.7, 43.3, 53.5 degrees in the enlarged Figure 4, the average measured


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relative FWHM values were 0.69, 0.40 and 0.36 cm for the core, core-shell and core-shell-shell nanoparticles which is consistent with the Scherrer equation and the TEM results. This information provided the theoretical basis to assume that the volume ratios of these ScNPs were similar to mole ratios.

Figure 4. XRD patterns of core NaLuF4:Gd(35%),Eu(15%), core-shell NaLuF4:Gd(35%), Eu(15%)@NaLuF4:Gd(40%) and core-shell-shell NaLuF4:Gd(35%),Eu(15%)@NaLuF4: Gd(40%)@NaLuF4:Gd(35%),Tb(15%), in comparison with JCPDS No.16-0334 β-NaYF4. From the TEM images we were able to estimate the volumes of the core and shell in these ScNPs according to a literature publised method with some modifications39 (Method S1, Supporting information). At the added 1:1:2 mole ratio for the core:middle shell:outer shell starting material mixtures, the core:middle shell:outer shell volume ratio was found to be about 1:0.78:1.30 after the reaction and purification, indicating that the loading efficiency was gradually reduced as the number of shells was increased from one to two. A unique method to compare the wt% and mole% compositions calculated by using the morphological images and the inductively coupled plasma (ICP) elemental analysis data of the core, core-shell and coreshell-shell ScNPs can now be developed. Assuming these ScNPs are spherical and the volume ratios are similar to mole ratios, the theoretical (based on starting reaction mole% data), calculated (based on TEM images) and experimental (based on ICP data) weight% and mole% compositions of Lu3+, Gd3+, Eu3+ and Tb3+ in the core, core-shell and core-shell-shell ScNPs are listed in Table 1. Detailed examples of calculations are also shown in Method S1 (Supporting information). It is noted that the differences between TEM estimated and the ICP data determined wt% and mole% for Lu3+, Gd3+, Eu3+ and Tb3+ are +1.7%, -2.0%, +1.4% and -1.0%, respectively. These discrepancies may result from the difficulties of handling small quantities of the hygroscopic starting materials as well as the errors due to ICP determinations.40 Table 1. The theoretical (based on starting reaction mole% data), calculated (based on TEM images) and experimental (based on ICP data) wt% and mole% compositions of Lu3+, Gd3+, Eu3+ and Tb3+ in the core, core-shell and core-shell-shell ScNPs.


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ScNPs

core

Compositions

Theoretical calculation

Page 10 of 25

core-shell

wt %

mol %

wt %

mol %

wt %

mol %

Lu

52.9%

50.0%

57.8%

55.0%

55.2%

52.5%

Gd

33.3%

35.0%

35.4%

37.5%

34.2%

36.3%

Eu

13.8%

15.0%

6.8%

7.5%

3.4%

3.7%

7.2%

7.5%

Tb

Calculation from TEM images

Lu

52.9%

50.0%

57.2%

54.4%

55.8%

53.1%

Gd

33.3%

35.0%

35.1%

37.2%

34.5%

36.6%

Eu

13.8%

15.0%

7.7%

8.4%

5.5%

6.0%

4.2%

4.4%

Tb

Experimental from ICP data

core-shell-shell

Lu

50.9%

48.0%

55.5%

52.7%

54.1%

51.4%

Gd

34.6%

36.3%

36.3%

38.4%

36.6%

38.6%

Eu

14.5%

15.7%

8.2%

8.9%

4.2%

4.6%

5.2%

5.4%

Tb

3.2. Photoluminescence (PL) and X-ray excited luminescence (XL) of ScNPs. The PL spectra of the core and core-shell-shell ScNPs were measured at room temperature, as shown in Figure S1 (Supporting information). The emission spectrum of the core ScNP shows the 590 nm (5D0→7F1), 614 nm (5D0→7F2) and 695 nm bands that are attributed to Eu3+. On the other hand, the emission spectrum of the core-shell-shell ScNP shows additional 488 nm (5D4→7F6) and 543 nm (5D4→7F5) bands due to doped Tb3+ in the second shell. Figure 5(a) shows the emission spectra of the NaLuF4:Gd,Eu@NaLuF4:Gd@NaLuF4: Gd,Tb core-shell-shell ScNPs irradiated by UV (λex = 273 nm) and X-ray lights. Both spectra exhibit bands centered at 488 nm (5D4→7F6) and 543 nm (5D4→7F5) due to Tb3+, and 590 nm (5D0→7F1), 614 nm (5D0→7F2) and 695 nm (5D0→7F4) due to Eu3+. This confirms that X-ray irradiation can also generate emission spectrum similar to that irradiated by UV light. Note that the X-ray-stimulated luminescence peak intensities in our work exhibited relatively lower signalto-noise ratios which could be due to that the X-ray power density applied under normal conditions was likely much lower than that of the UV excitation source (5 mW/cm2). We are in the process to understand the problem in more detail and will try to optimize the situation at a later stage for practical medical applications. For the initial X-ray induced optical imaging study, a home-made fuorescence/computed tomography (FT-CT) instrument was used.41 Figure 5(b) shows that the core-shell-shell ScNPs have luminescence properties similar to that of NaGdF4:Eu16 and reasonably good imaging signals could be obtained within 30s.


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Figure 5. (a) Emission spectra of the core-shell-shell ScNPs excited at λex = 273 nm and by Xray. X-ray excitation source: 50 keV, 0.5 mA; detector slit width: 10 nm; temp: 28.75 ℃; dwell time: 1 s. UV excitation source: 273 nm; detector slit width: 5 nm; temp: 28.75 ℃; dwell time: 0.1 s. The unit of Y axis = arbitrary unit of photon counts. (b) X-ray induced luminescence using a homemade FT-CT instrument: positions of the NaGdF4:Eu(15%) ScNPs (left, red arrow) and our core-shell-shell ScNPs (left, blue arrow), images obtained after 10s X-ray irradiation (middle) and images obtained after 30s X-ray irradiation (right). Previously, it was reported that the cubic nanomaterial BaYF5:Eu3+ prepared by a similar method gave relatively good luminescence images in an animal model when excited by X-ray with emissions at 614 nm and 695 nm, and its resolution was better than PET.42 In another paper, it was shown that the X-ray excited luminescence of the hexagonal NaGdF4:Eu3+ was better than the cubic BaGdF5:Eu3+and BaYF5:Eu3+.16 Because the X-ray excited luminescence imaging equipment is not commercially available and the homemade ones at different laboratories with different specifications show variable qualities, our imaging measurements could only use the one accessible to us which may not be as good as others under development.43,44 The EMCCD detector of our homemade X-ray excited fluorescence/computed tomography (FT-CT) instrument could only work in the visible (i.e. covering emissions at the 614 nm and 695 nm) but


11


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not NIR light region. Note that the Eu3+ emission at 695 nm is closer to the NIR region and might be advantageous for optical imaging to improve the penetration depth. Thus, although the resolution and signal-to-noise ratios of our X-ray excited FT-CT instrument and image reconstruction softwares need further improvement,41 we believe that we have successfully demonstrated that it is possible to use a single X-ray excitation source and well-designed nanocomposites such as ours to perform both photodynamic therapy (vide infra) and luminescence imaging at the proof-of-principle stage. 3.3. Quantitation of loaded photosensitizer RB and its release in solutions of different pH. The amount of loaded RB onto the core-shell-shell ScNPs was estimated by subtracting the unreacted RB in the reaction supernatant after centrifugation from the total added RB using equation 1. A standard RB calibration curve was prepared to determine the unreacted RB (Figure S2, Supporting information). Photosensitizer loading(%) =

! " !# !#

× 100%

(1)

It was found that at the added 2 mg RB per 10 mg ScNPs in triplicates, the amounts of RB in the supernatants were 1.037, 1.019, and 1.056 mg for the ScNP-PAH nanoparticles, and 0.0879, 0.100, and 0.107 mg for the ScNP-mSiO2-PAH nanoparticles. This indicated that 9.63 ± 0.18 and 19.02 ± 0.09 (wt%) of RB were loaded onto the ScNP-PAH and ScNP-mSiO2-PAH nanoparticles, respectively. The higher RB loading amount of the latter is consistent with the mSiO2 modification on the surface of ScNP-mSiO2-PAH nanoparticles which provided more pores and PAH molecules to load RB. On the other hand, only half of the RB was loaded onto the ScNP-PAH nanoparticles and it was speculated that there might be less PAH molecules on the surface of the ScNP-PAH to interact with RB as compared to the ScNP-mSiO2-PAH nanoparticles. Unfortunately, although the ScNP-mSiO2-PAH-RB contained more loaded RB, after further modification with PEG-FA, its 1O2 generation efficiency was much inferior to that without the mSiO2 layer (vide infra). The TEM image and DLS analysis of the ScNP-PAH-RB-PEG-FA nanocomposites are shown in Figure S3 (Supporting information). It was seen that after the coating with PAH-RBPEG-FA, the TEM image of the nanocomposites showed that the shapes and average size were roughly maintained, but they tended to form aggregates with time, similar to those previously reported.13 A mean diameter of 171.0 ± 19.1 nm was obtained by DLS analysis. To estimate the stability of the loaded RB onto the ScNPs in solution, a photosensitizer release test was performed in buffer solutions with different pH. Because the ScNP-mSiO2-PAHRB-PEG-FA nanoparticles were ineffective in generating 1O2 molecules, only the ScNP-PAHRB-PEG-FA nanoparticles without the mSiO2 layer were tested. The ScNPs were dispersed in MES (pH 6.5), PBS (pH 7.4) and carbonate-bicarbonate (pH 8.6) buffer solutions (2 mL) for 24 h and then centrifuged, and re-dispersed in separate buffer solutions. The spectra of RB were measured at t = 0 and t = 24 h for the three solutions and the absorbance differences between t = 0 and t = 24 h at 549 nm were used to calculate RB release percentages after 24 h. The results showed that RB losses were 37% (pH 6.5), 36.6% (pH 7.4) and 55% (pH 8.6), respectively. Because the pKa values of RB are 1.89 and 3.93,45 in the pH 6.5-8.6 range, RB is primarily


12

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in the 2- charged form and its interaction with the ScNPs-PAH are mainly electrostatic in nature. On the other hand, the ScNP-PAH-NH3+ has an effective pKa value close to 8.5,46 thus at pH 8.6, relatively more than half of the amine functional groups of PAH are deprotonated which results in less positive charges and more release of RB as compared to those at more acidic pH 6.5 and 7.4. This makes our ScNPs suitable for tumor PDT because tumor microenvironment is often more acidic than normal tissue. 3.4. Zeta potential measurements for ScNPs with different surface modifications. The zeta potential would be different when the surface of ScNPs was modified.40,47 Figure 6(a) shows that the zeta potential of the positively charged ScNP-PAH (+67 mV) due to PAH-NH3+ functional groups is changed to negatively charged -30.8 mV for ScNP-PAH-RB-PEG-FA due to the modifications with nagatively charged PEG-folate and RB. Figure 6(b) shows that the zeta potentials of the surface modified ScNPs with mSiO2 and PAH are positively charged, and those of CTAC-removed and PEG-FA modified are negatively charged. Specifically, after mSiO2 coating, the zeta potential was positive charged (i.e. +28.2 mV) due to the pore template CTAC. When the template CTAC was removed, a negative zeta potential was observed, i.e. -26.5 mV, due to silanol dissociation to form Si-O-Si-O- groups on the surface.

Figure 6. Zeta potential data of ScNPs: (a) ScNP-PAH and ScNP-PAH-RB-PEG-FA; and (b) ScNP-mSiO2, ScNP-mSiO2 CTAC removed, ScNP-mSiO2-PAH and ScNPs-mSiO2-PAH-RBPEG-FA. 3.5. Lifetime measurement. To understand the efficiency of energy transfer,34,47,48, we carried out lifetime (luminescence decay curve) measurements of the emission peaks at 543 nm for the Tb3+ 5D4→7F5 transition in the core-shell-shell ScNPs before and after loading of RB (Figure 7, λex = 355 nm).


13


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Figure 7. Exponential decay curves of the Tb3+ 5D4→7F5 transition (λex=355 nm, λem=543 nm) in the core-shell-shell ScNPs before & after loading of RB: (a) ScNP@mSiO2-PAH and (b) ScNPPAH. Table 2 lists the lifetime data obtained by fitting the curves in Figure 7 to the following diexponential decay equation (Eq. 2): It = I1exp(-t/τ1) + I2exp(-t/τ2)

(2)

where It is the total luminescence intensity at time t, I1 and I2 are the component intensities at t = 0, and τ1, and τ2 are the lifetimes for each component. Note that there are at least three possible but not mutually exclusive explanations available for these luminescence decay models with more than one decay components.34 One is that the probability for the non-radiative decay is different between the LnIII ions at or near the surface and the LnIII ions in the core of the NPs. The second is that inhomogeneous distribution of the dopant LnIII ions in the host material leads to variations in local doping concentrations. The third is that there is energy transfer from LnIII (e.g. Tb3+) donors to LnIII (e.g. Eu3+) acceptors. In hexane without the water molecules quenching, the Tb3+ ions in the ScNP had a τ value 4569 µs. After surface modification with mSiO2 and/or PAH, two lifetime components were found for the Tb3+ ions in aqueous solutions. The shorter one was attributed to those near the particle surface which could be affected by water quenching. The longer one was attributed to those more inside without water quenching. In the cases of energy transfer from the donor Tb3+ ion to the acceptor rose bengal (RB), to make a simplified comparison, the average lifetimes (τav) of these nanoparticles were calculated using Eq. 3,34,49 and the results are also listed in Table 2: τav = (I1τ12+I2τ22)/(I1τ1+I2τ2)

(3)

Table 2. lifetime data of ScNP in hexane, core-shell-shell ScNP@mSiO2-PAH and ScNP@PAH with and without RB loading in aqueous solutions. ScNPs

I1

' 1 (µS)

ScNP in hexane

4378±6 (100%)

4569±6

ScNP@mSiO2-PAH

886±34 (15%)

1429±52

1788±37 (85%)

ScNP@mSiO2-PAH w RB

78±2 (100%)

610±37

N/A

I2

X2

' av (µS)

1.072

4569

3994±39

1.078

3525

N/A

1.042

610

' 2 (µS)


14

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ScNP@PAH

864±138 (2.5%)

1324±171

10136±135 (97.5%)

4331±47

1.042

4243

ScNP@PAH w RB

N/A

N/A

N/A

N/A

N/A

29

Figure 7 (left) shows that for the NaLuF4:Gd,Eu@Gd,Lu@Gd,Lu,Tb@mSiO2-PAH without RB loading, the τav lifetime was determined to be 3525 µs and it decreased to 610 µs after the loading of RB. Figure 7 (right) shows that for the NaLuF4:Gd,Eu@Gd,Lu@Gd,Lu,Tb@PAH without RB loading, the τav lifetime was determined to be 4243 µs. After the loading of RB, the lifetime of the NaLuF4:Gd,Eu@Gd,Lu@Gd,Lu,Tb@PAH was dramatically decreased and the signal was too weak to fit, we estimated the lifetime to be 29 µs. The shortening of the τav lifetime is indicative of energy transfer from Tb3+ to RB which could be used to calculate FRET efficiency (ηET) by the Eq. 4: ηET = 1 – τobs/τ0

(4)

where τobs is the lifetime of the ScNPs with RB, τ0 is the lifetime of nanoparticles without RB. The calculated FRET efficiency values are 82.7% for the ScNP@mSiO2-PAH and 99.3% for ScNP@PAH, respectively. The results indicate that the energy transfer is less efficient with an extra layer of mSiO2 as compared to that directly loading with rose bengal (RB). This is very likely due to the presence of the thick 25 nm mSiO2 shell which separates the donor from the acceptor further and reduce the FRET efficiency. 3.6. X-ray excited singlet oxygen generation test. The 1O2 generation by the core-shellshell ScNP-PAH-RB-PEG-FA and ScNP-mSiO2-PAH-RB-PEG-FA without and with the mSiO2 layer under X-ray excitation was tested by employing ABDA which is a commercial molecular probe for the detection of 1O2 production.30,50 In the presence of 1O2, ABDA is oxidized and accompanied by the quenching of its fluorescence at 407 nm. Figure 8 shows that significantly extra percentages of 1O2 were produced via FRET process for the core-shell-shell ScNP-PAHRB-PEG-FA without the mSiO2 layer as compared to that of the ABDA control up to 10 Gy Xray radiation (P=0.025). On the other hand, for the core-shell-shell ScNP-mSiO2-PAH-RB-PEGFA with the mSiO2 layer, there was no significant amount of 1O2 produced up to 5 Gy X-ray radiation. Further radiation was stopped because the 1O2 produced at higher X-ray doses would be mainly due to the oxidation of ABDA. Although previously it was shown that the FRET between Tb3+ and RB was still 82.7%, apparently the conversion of the energy from RB to generate 1O2 was hampered due to the presence of the mSiO2 layer, regardless the loaded amount of the RB was almost twice that without the mSiO2 layer. This result implied that the average distance between RB and the Tb3+ in the ScNPs outer shell for the photochemical reaction to generate 1O2 molecules is very important for effective PDT. In the paper by Wang, D., et al.,28 it was shown in the NaYF4:Yb,Ho@NaYF4:Nd @NaYF4 core-shell-shell nanostructures, the emission energy transfer efficiency to generate 1O2 was affected by the distance between the Ho3+ emitter and RB acceptor. The optimum distance was 1.6 nm which was far shorter than the 25 nm thickness of mSiO2. Further optimization studies might be needed at later development stage to examine how the shell and mSiO2 layer thicknesses affect the efficiency on the generation of 1O2 molecules.


15


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Thus, only the core-shell-shell ScNP-PAH-RB-PEG-FA without the mSiO2 layer was used for further cellular uptake, cytotoxicity and PDT studies.

Figure 8. The 1O2 generation test by the core-shell-shell ScNP-PAH-RB-PEG-FA and ScNPmSiO2-PAH-RB-PEG-FA under X-ray irradiation employing ABDA. ABDA fluorescence decay at 407 nm is plotted against X-ray exposure dose (Gy). 3.7. Cellular uptake studies. Cellular uptake of the core-shell-shell ScNP-PAH-RB-PEGFA was investigated with MDA-MB-231 and MCF-7 cancer cells utlizing the 543 nm green light emitted by Tb3+ and laser confocal imaging technique (Figure 9). Compared with that of the control without the ScNPs (i.e. top images), green photoluminescence was observed and distributed across the cytoplasm but not in the nuclei of the cells (i.e. bottom images), indicating that ScNP-PAH-RB-PEG-FA nanoparticles were internalized by cells through endocytosis,51 which could be facilitated by the folate functional groups and electrostatic interactions between the particles and the cell membranes. These results provided evidence that our ScNPs could be useful as probes for laser confocal imaging. Note that the confocal imaging results should not be used rigorously to compare quantitatively the uptake amounts of the nanocomposites by the MAD-MB-231 and the MCF-7 cells, because the nature and number of cells as well as instrument settings were different to obtain better images, as pointed out by a number of reports previously.52,53


16

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Figure 9. Confocal cell images of the core-shell-shell ScNP-PAH-RB-PEG-FA incubated with (a) MDA-MB-231 and (b) MCF-7 cancer cells. Green fluorescence is from Tb3+ of ScNPs and blue fluorescence is from nuclear counterstaining with hochest-33342 (scale bars, 50 µm). 3.8. Dark cytotoxicity and X-ray excited photodynamic therapy (PDT) test. The MDAMB-231 and MCF-7 cells were incubated with different concentrations of the core-shell-shell ScNP-PAH-RB-PEG-FA for 24 hours in dark and the cell viability were measured using CCK-8 kit. The results were shown in Figure 10(a). Compared with that of the control without ScNPs, it was found that 82.5% cells survived even at a high concentration of 400 µg/mL for the MDAMB-231 cells, and the survival rate of MCF-7 cells was even better. This suggested the good biocompatibility of our ScNP-PAH-RB-PEG-FA nanocomposite in 24 hrs.

Figure 10. (a) Cell viability tests of MDA-MB-231 (blue) and MCF-7 (red) cancer cells incubated with different concentrations of the core-shell-shell ScNP-PAH-RB-PEG-FA in dark. (b) In vitro X-ray induced PDT on MDA-MB-231 (yellow) and MCF-7 cells (orange). Cell viabilities of MAD-MB-231 and MCF-7 cells treated with 50 µg/mL core-shell-shell ScNPPAH-RB-PEG-FA for 24 h followed by 1, 3, 5 Gy of X-ray radiation doses. The control groups were those with X-ray irradiation without the core-shell-shell ScNP-PAH-RB-PEG-FA nanocomposite (***P < 0.0001). Figure 10(b) shows that the cell viability of MDA-MB-231 and MCF-7 treated with 50 µg/mL core-shell-shell ScNP-PAH-RB-PEG-FA nanocomposites at different X-ray radiation


17


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doses. It was observed that both cell viabilities were significantly decreased as compared to those without the ScNPs starting at the X-ray irradiation of 1 Gy. Note that the irradiation dose rate of our RS-2000 equipment was set at 3.5 Gy/min. A dose of 1 Gy required only 17 second irradiation and which gave moderate initial therapy efficacy. This result was already similar to that of the previous report using the CeF3-VP material with a 6 Gy dose (vide infra). At a radiation dose of 5 Gy, 31% MDA-MB-231 and 21% MCF-7 cells were killed as compared to that of the control. Further increase of the X-ray radiation doses up to 5 Gy caused limited or no additional decreases of the cell viabilities, which was similar to that reported previously,54 and was probably because the concentration of local triplet 3O2 molecules was diminished due to longer X-ray irradiation around the cancer cells. Nevertheless, this may be regarded as a strong evidence to show that the main damage of the cells was caused by X-ray excited PDT employing the newly synthesized core-shell-shell ScNP-PAH-RB-PEG-FA nanocomposites, instead of Xray radiation itself. It is interesting to observe that the X-ray excited PDT results showed that the viability was slightly greater for MCF-7 cells than MDA-MB-231 cells at higher X-ray doses studied. This was reasonable because the MDA-MB-231 cells expressed more folate receptors55 and the uptake of nanocomposites would be more than that of the MCF-7 cells, which would result in more MDA-MB-231 cells been killed, if other factors were not considered. In addition, it was reported that the ER-positive MCF-7 breast cancer cells lack Caspase-3 proteins which is an important factor in apoptosis.56 This would make MCF-7 cells be more resistant to PDT as well as other chemotherapy than the MDA-MB-231 cells.57 Table 3 shows the preliminary comparisons of PDT cell viabilities using different materials and X-ray dosages. Except the one employing the SrAl2O4:Eu2+-MC540 material, the core-shellshell ScNP-PAH-RB-PEG-FA used in the current study showed similar or greater cytotoxicities as compared to those of other materials using X-ray radiation at equivalent dosages. Although more thorough comparisons of the PDT efficacies of these materials could be performed under better controlled experimental conditions and equipment used, the potentially additional fluorescence imaging capability provided by our nanocomposites using a single X-ray excitation source is of certain advantages as theranostics. Table 3. Preliminary comparisons of PDT cell viabilities using different materials and X-ray dosages. Material Y2O3-psoralen, 95 µg/mL LiYF4:Ce@ZnO, 50 µg/mL SrAl2O4:Eu2+-MC540, 50 µg/mL CeF3-VP(verteporfin), 16 µg/mL Core-shell-shell ScNP-RB, 50 µg/mL

X-ray dosage 2 Gy 2-6 Gy 1 Gy 1-6 Gy 1-5 Gy

Cell viability 79% (2 Gy) 85% (2 Gy), 68% (6 Gy) 40% (1 Gy) 90% (1 Gy), 70% (6 Gy) 80% (1 Gy), 70% (5 Gy)

Date & Ref. 2011,58 2015,14 2015,15 2016,54 This study

Finally, although we have made some progress to demonstrate the possibility of single source X-ray excited photodynamic therapy and luminescence imaging, there are still many factors to consider to make it feasible for clinical applications, including the improvement of Xray induced PDT effect. This could be done by improving the carrier/photosensitizer efficiency using different materials, better formulation and loading of the photosensitizers, increasing dose,


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and optimization of instrument parameters for specific use. 4. Conclusions In this work, an X-ray excited core-shell-shell ScNP-PAH-RB-PEG-FA nanocomposite has been constructed as the first example for dual photodynamic therapy and luminescence diagnostic imaging to image and kill MDA-MB-231 & MCF-7 cancer cells with low dark cytotoxicity and efficient photocytotoxicity. Owing to the unique scintillating property and X-ray attenuation ability of the NaLuF4:Gd,Eu@Gd,Lu@Gd,Lu,Tb nanoparticles, and with the use of appropriate photosensitizers such as rose bengal (RB) and biomarker derivatives, the integral FRET system could generate 1O2 molecules and be applied for targeted PDT of deep-tissue tumor. Further work is needed to optimize the conversion of the X-ray to visible or near infrared lights with high signal-to-noise ratios by this kind of ScNPs for better imaging quality and PDT efficiency, together with the successful development of the X-ray excited FT-CT instrument. It is hoped that this approach may eventually be useful as a powerful platform for image-guided theranostics against cancer in the future. ASSOCIATED CONTENT Supporting Information Available: Geometric TEM morphological and ICP data analysis of core, core-shell and core-shell-shell ScNPs (Method S1), the emission spectra of NaLuF4:Gd(35%),Eu(15%) and NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%)@NaLuF4: Gd(35%),Tb(15%) ( Figure S1), the calibration curve of rose bengal (Figure S2), and the TEM image and DLS analysis of the ScNP-PAH-RB-PEG-FA nanocomposites (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. (C. A. Chang). Author Contributions Chang-Chieh Hsu and Syue-Liang Lin contributed equally to this paper. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was supported by the Ministry of Science and Technology of Taiwan (NSC1022627-E-010-001, NSC102-2627-M-010-001, MOST103–2627-M-010–001, MOST104-2627-M010-001, MOST105-2113-M-010-001, MOST106-2113-M-010-006), Veterans General Hospitals University System of Taiwan Joint Research Program (VGHUST104-G7–4–1),


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Taiwan (ROC) and the Professor Tsuei-Chu Mong Merit Scholarship of National Yang-Ming University. Thanks are also due Prof. H. K. Chiang, Ms. P.-A. Lo, Prof. J.-C. Chen and Mr. S. C. Jin for the help with the use of the home made FT-CT facility. ABBREVIATIONS ScNPs, scintillator nanoparticles; FT-CT, Fluorescence Tomography Computed Tomography; PDT, photodynamic therapy; ROS, reactive oxygen species; MTCP, meso-tetra(4-carboxyphenyl) porphine; FRET, fluorescence resonance energy transfer; RB, rose bengal; CTAC, cetyltrimethylammonium chloride; TEOS, tetraethyl orthosilicate; ABDA, 9,10-anthracenediylbis(methylene)-dimalonic acid; PAH, poly(allylamine); DLS, Dynamic lighting scattering; XL, X-ray excited luminescence; REFERENCES (1) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3 (5), 380-387. (2) Lin, L.; Xiong, L.; Wen, Y.; Lei, S.; Deng, X.; Liu, Z.; Chen, W.; Miao, X. Active targeting of nano-photosensitizer delivery systems for photodynamic therapy of cancer stem cells. J. Biomed. Nanotechnol. 2015, 11 (4), 531-554. (3) O’Connor, A. E.; Gallagher, W. M.; Byrne, A. T. Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy. Photochem. Photobiol. 2009, 85 (5), 1053-1074. (4) Punjabi, A.; Wu, X.; Tokatli-Apollon, A.; El-Rifai, M.; Lee, H.; Zhang, Y.; Wang, C.; Liu, Z.; Chan, E. M.; Duan, C. Amplifying the red-emission of upconverting nanoparticles for biocompatible clinically used prodrug-induced photodynamic therapy. ACS Nano 2014, 8 (10), 10621-10630. (5) Fan, W.; Huang, P.; Chen, X. Overcoming the Achilles' heel of photodynamic therapy. Chem. Soc. Rev. 2016, 45 (23), 6488-6519. (6) Chen, N.-T.; Tang, K.-C.; Chung, M.-F.; Cheng, S.-H.; Huang, C.-M.; Chu, C.-H.; Chou, P.T.; Souris, J. S.; Chen, C.-T.; Mou, C.-Y. Enhanced plasmonic resonance energy transfer in mesoporous silica-encased gold nanorod for two-photon-activated photodynamic therapy. Theranostics 2014, 4 (8), 798-807. (7) Wang, C.; Tao, H.; Cheng, L.; Liu, Z. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles. Biomaterials 2011, 32 (26), 6145-6154. (8) Lucky, S. S.; Muhammad Idris, N.; Li, Z.; Huang, K.; Soo, K. C.; Zhang, Y. Titania coated upconversion nanoparticles for near-infrared light triggered photodynamic therapy. ACS Nano 2015, 9 (1), 191-205.


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