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Nov 8, 2016 - OECM-1 oral cancer cells. Consequently, UCP@SiO2:MB-NRs-FA could highly produce ROS and undergo efficient PDT in vitro and in vivo...
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Plasmon-Enhanced Photodynamic Cancer Therapy by Upconversion Nanoparticles Conjugated with Au Nanorods Chieh-Wei Chen,† Yung-Chieh Chan,‡ Michael Hsiao,*,‡,⊥ and Ru-Shi Liu*,†,‡,§ †

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan Genomics Research Center, Academia Sinica, Taipei 115, Taiwan § Department of Mechanical Engineering and Graduate Institute of Manufacturing Technology, National Taipei University of Technology, Taipei 106, Taiwan ⊥ Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan ‡

S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) based on photosensitizers (PSs) constructed with nanomaterials has been widely applied to treat cancer. This therapy is characterized by an improved PS accumulation in tumor regions. However, challenges, such as short penetration depth of light and low extinction coefficient of PSs, limit PDT applications. In this study, a nanocomposite consisting of NaYF4:Yb/Er upconversion nanoparticles (UCPs) conjugated with gold nanorods (Au NRs) was developed to improve the therapeutic efficiency of PDT. Methylene blue (MB) was embedded in a silica shell for plasmon-enhanced PDT. UCPs served as a light converter from near-infrared (NIR) to visible light to excite MB to generate reactive oxygen species (ROS). Au NRs could effectively enhance upconversion efficiency and ROS content through a localized surface plasmon resonance (SPR) effect. Silica shell thickness was adjusted to investigate the optimized MB loading amount, ROS production capability, and efficient distance for plasmon-enhanced ROS production. The mechanism of plasmon-enhanced PDT was verified by enhancing UC luminescence intensity through the plasmonic field and by increasing the light-harvesting capability and absorption cross section of the system. This process improved the ROS generation by comparing the exchange of Au NRs to Au nanoparticles with different SPR bands. NIR-triggered nanocomposites of UCP@SiO2:MB-NRs were significantly confirmed by improving ROS generation and further modifying folic acid (FA) to develop an active component targeting OECM-1 oral cancer cells. Consequently, UCP@SiO2:MB-NRs-FA could highly produce ROS and undergo efficient PDT in vitro and in vivo. The mechanism of PDT treatment by UCP@SiO2:MB-NRs-FA was evaluated via the cell apoptosis pathway. The proposed process is a promising strategy to enhance ROS production through plasmonic field enhancement and thus achieve high PDT therapeutic efficacy. KEYWORDS: upconversion nanoparticle, gold nanorod, localized surface plasmon resonance, plasmonic enhancement, near-infrared light triggering, photodynamic therapy

1. INTRODUCTION Lanthanide-doped upconversion nanoparticles (UCPs), which involve a nonlinear optical process, present low cytotoxicity, long excitation wavelength, and low autofluorescence background. As such, UCPs are promising nanomaterials (NMs) in biological applications, such as biosensors, bioimaging, siRNA delivery, and cancer treatment.1−5 UCPs are fabricated by doping a small amount of sensitizers and activators in an inorganic nanocrystalline host. The complex emission of upconversion (UC) processes occurs through the transfer of energy between sensitizers, such as Yb3+, which is the most commonly used sensitizer, and activators, such as Er3+, Tm3+, and Ho3+. Given this distinct ability to transfer energy, UCPs can provide a remotely phototriggered response system, which can be excited by a 980 nm laser; consequently, multicolor © 2016 American Chemical Society

emission occurs in the UV and visible regions by doping various activators for drug release or intracellular signal control.6,7 UCPs are key light conductors for photodynamic therapy (PDT) to kill malignant cells. PDT involves the reaction of an excited photosensitizer (PS) with surrounding oxygen molecules or substances to generate reactive oxygen species (ROS); thus, PDT exhibits a high capability for cell destruction.8,9 ROS production is a major concern in PDT. However, poor penetration depth in an organism’s tissue and low-efficiency ROS production by a source under the visible light excitation of sensitizers have caused limitations to practical PDT applicaReceived: June 27, 2016 Accepted: November 8, 2016 Published: November 8, 2016 32108

DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119

Research Article

ACS Applied Materials & Interfaces

lower than that of Au NRs. Thus, the conjugation of ICG and Au NRs as one nanoplatform enlarges the absorption cross section of ICG and promotes an efficient ROS generation caused by the coupling effect with Au NRs. Li et al. verified that this nanoplatform can be used in PDT and PTT because Au NRs can undergo photothermal conversion via LSPR effect. We aimed to develop a new type of nanocomposites based on UCPs that can remarkably convert light for PDT. We also utilized the plasmonic field to improve the ROS production. To the best of our knowledge, this study is the first to propose this approach. Studies have focused on enhancing UCL because plasmonic field can also affect the behavior of the UC process through surface-plasmon-coupled emission (SPCE) or local field enhancement (LFE) effect by increasing the emission rate or excitation rate, respectively.18−20 In our study, nanocomposites were fabricated by coating a core/shell structure of UCP around a silica shell and directly conjugated with Au NRs on the surface. The PS of methylene blue (MB) was embedded inside the silica shell to increase photostability and prevent drug leakage. The silica shell was also examined to determine the optimized effective distance between Au NRs and MB and to enhance plasmonic field. The nonoverlapping LSPR band of Au nanoparticles (NPs) was modified to identify the enhanced effect resulting from the overlapping interaction between the absorption spectrum of MB and plasmonic resonance. Finally, the nanocomposite of UCP@SiO2:MBNRs was modified with folic acid (FA) to actively target OECM-1 oral cancer cells and to achieve high PDT efficacy in vitro and in vivo. PDT for the cell apoptosis pathway was investigated through staining to analyze the mitochondrial membrane potential (MMP, ΔΨm) and identify caspase 3 activity. The results indicated that the proposed approach can be applicable for PDT in vitro and in vivo.

tions. The therapeutic efficiency of PDT is enhanced by directly shifting the excitation source to the NIR light of 980 nm. This process shows an excellent penetration ability because water, hemoglobin, and melanin inside an organism absorb energy below 700 nm and above 1100 nm.10 PDT efficiency has been improved by using several approaches. Idris et al.11 presented an intuitive concept of combining two PSs inside a UCP nanoplatform. They also coated a UCP on a mesoporous silica shell to load PS, in which UCPs doped with Yb3+ and Er3+ emit red (∼660 nm) and green (∼540 nm) luminescence to excite zinc(II) phthalocyanine and merocyanine 540, respectively, and thus increase ROS production. They observed that the amount of ROS generated by the coloading of PSs in UCP@mSiO2 nanoplatforms is 2-fold higher than that produced by the loading of one PS under irradiation of 980 nm laser for 80 min. Wang et al.12 developed a simple structure to enhance the transfer of energy from UCPs to PS. They claimed that the efficiency of Förster resonance energy transfer between a donor and an acceptor is the key problem that reduces the distance between a light source and a PS. This characteristic can directly affect the enhancement of ROS production. Thus, PSs surrounding UCPs are directly modified and the protective ligands of oleic acid (OA) are removed. Thus, ligand-free UCPs are produced. Therapeutic efficacy is also improved with the simultaneous application of multiteratments, which result in a synergistic effect. Chen et al.13 demonstrated a coloaded drug system based on the UCP modification of bovine serum albumin (BSA) protein. This protein can be used to adsorb PSs, such as rose bengal (RB), and a NIR-absorbing dye, such as IR825, for PDT and photothermal therapy (PTT), respectively. UC red light emission can be further applied for bioimaging without any influence from other substances because RB can only absorb the energy at green light (∼540 nm), and IR825 is irradiated by another 808 nm laser. PDT based on UCP systems has been improved primarily by enhancing the property of multicolor emission or the efficiency of energy transfer to PSs. Nevertheless, poor ROS production from sensitizers in a UCP system has been rarely promoted. In this study, ROS production was enhanced by a plasmonic field, which resulted from noble metallic NMs, such as silver and gold (Au) NMs. Noble metallic NMs exhibit a distinct SPR originating from numerous free conductive electrons on surfaces. These electrons easily interact with incident light and subsequently cause a collective oscillation at a specific frequency.14 Localized SPR (LSPR) is generated by particles smaller than the incident light. Zhang et al.15,16 investigated the mechanism of metal-enhanced singlet oxygen generation and discovered that RB absorption is enhanced by placing RB on identical glasses or silver films because of the coupling effect between the different distances from the silver film to RB. This effect is related to the metal-enhanced fluorescence or phosphorescence because a photoinduced electronic field can also enhance the fluorescence or phosphorescence emission. The effective distance is also identified by sequentially coating various thicknesses of SiOx layer. Maximum enhancement is observed at approximately 0.5 nm, and no enhancement is observed above 10 nm. However, Zhang et al. conducted a simulation or extracellular demonstration of this phenomenon. Li et al.17 provided an evidence supporting plasmonic enhancement by Au NMs. NIR-triggered Au nanorods (NRs) conjugated with indocyanine green (ICG) induce the plasmonic field and enhance the ROS production from ICG. The extinction coefficient of ICG at 780 nm is considerably

2. EXPERIMENTAL WORK 2.1. Chemicals and Materials. MB (98%), IGEPAL CO-520, TEOS (98%), APTES (98%), NHS, EDC, and FA were purchased from Sigma-Aldrich. All chemicals were used as received without further purification. Hochest 33342 was purchased from Jackson ImmunoResearch Laboratories. The water used was of reagent-grade and produced using a Milli-Q SP ultrapure-water purification system from Nihon Millipore Ltd. (Tokyo). 2.2. Preparation of UCP@SiO2:MB. NaYF4:Yb,Er NPs and UCP@SiO2:MB were synthesized using our previous procedure.21 UCNP (1 mL, 10 mg/mL) was added into the mixture of CO-520/ cyclohexane solution. Afterward, 200 μL of MB (6 mM) was added into the solution, and the mixture was mixed well by stirring. Ammonia (30%, 100 μL) was added to the mixture after 2 h. A syringe pump was used to control the addition rate, and 40 μL of TEOS was added to the mixture. The mixture was sealed and stirred for 24 h. Ethanol was then added to precipitate the product, which was collected by centrifugation. The precipitate was washed with ethanol several times, and the product was finally dispersed in deionized water. 2.3. Detection of ROS in Aqueous Solution. 9,10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA) and 1,3-diphenylisobenzofuran (DPBF) were used to react with 1O2 to detect the ROS in the aqueous solution. UCP@SiO2, UCP@SiO2:MB, and UCP@SiO2:MBNRs-FA nanocomposites (1 mg/mL) in 1 mL of deionized water containing 10 μM of ABDA dye were used in a typical ABDA experiment. The mixture was then placed in a cuvette and stirred. The solution was irradiated with 1.5 W/cm2 continuous wavelength (CW) 980 nm laser for 60 min with 5 min intervals of “on” and “off” conditions beginning from time (t) = 0; however, the first measurement was performed after 10 min of irradiation. ROS production was confirmed via detecting the fluorescence emission of ABDA at 407 nm by excitation at 380 nm. The fluorescent spectra of 32109

DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic diagram of the preparation of upconversion nanoparticle (UCP)@SiO2:methylene blue (MB)-nanorods (NRs)-folic acid (FA). The silica shell was coated with reverse-phase microemulsion and modified FA and Au NRs through NHS/EDC coupling reaction and electrostatic force, respectively. 2.6. In Vivo PDT Treatment. Animal experiments were approved by the Institutional Animal Care and Utilization Committees of Academia Sinica (IACUC NO. 16-05-957). OECM-1 cells (5 × 106 cells/100 μL) were injected subcutaneously on the backside of mouse. UCP@SiO2-NRs-FAs (100 mg) with and without MB loading were intratumorally injected into the tumor when tumors had grown to 125 mm3. Irradiation with 150 mW/cm2 of 980 nm IR-laser was performed after 12 h incubation for 30 min under 5 min intervals of cooling down and irradiation. All tumors were then acquired after 1 week. The tumor sections were formalin fixed and paraffin embedded. The cross sections of tumor were stained using hematoxylin and eosin. All the staining tumor sections are shown using Leica Aperio AT2 scanner. 2.7. Detection of MMP. MMP was detected immediately after laser treatment using JC-1 staining process according to the manufacturer’s instruction. The culture medium of OECM-1 oral cancer cells that were exposed to 250 μg/mL nanocomposites (UCP@ SiO2:MB-FA and UCP@SiO2:MB-NRs-FA) for 12 h. Afterward, the cells were subjected to exposure with CW 980 nm laser at 1.5 W/cm2 for 30 min. Approximately 25 μg/mL JC-1 and 0.1 μg/mL DAPI were sufficiently added and used to cover the adhering cells. The fluorescent images of the cells stained with JC-1 and DAPI were promptly captured by the Leica TCS SP5 confocal microscope after excitations at 480, 540, and 350 nm. The high-membrane potential resulted in the JC-1 aggregation in the membrane, as shown by the strong red fluorescent signal (EX/EM = 540/590 nm), and low-membrane potential caused the monodispersed release of JC-1 to the cytosol, as shown by the strong green fluorescent signal (EX/EM = 480/520 nm). DAPI was used to mark the nucleus site. 2.8. Caspase 3 Activity Assay. Western blot analysis was performed as previously reported (Chan et al.).36 Cells were lysed with RIPA solution (150 nM NaCl, 1% NP-40, 50 mM Tris-Cl pH 8.0, 2 mM EDTA (pH 8.0), 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors) according to regular operation. Protein concentration was determined by Pierce BCA assay (Thermo, USA). The primary antibodies were diluted in 2% BSA/PBST buffer (137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 0.1% Tween 20; pH 7.4). Cell apoptosis was examined using anticaspase-3 that detected full-length caspase-3 (35 kDa) and the 17 kDa cleavage fragment

the mixture after NIR-laser irradiation were recorded by Fluoromax-3 (Horiba, Japan). UCP@SiO2:MB-NPs-FA and UCP@SiO2:MB-NRsFA nanocomposites (1 mg/mL) in 1 mL of deionized water containing 100 μM of DPBF dye were used in a typical DPBF experiment. This dye reacted irreversibly with 1O2 to reduce the intensity of the DPBF absorption band at approximately 420 nm. The solution was then placed in a cuvette and stirred. The solution was irradiated at 1.5 W/cm2 using CW 980 nm laser for 60 min. The absorption spectra of the mixture after NIR-laser irradiation were recorded by UV-1700 spectrophotometer (Shimadzu, Japan). 2.4. Detection of Intracellular ROS. Intracellular ROS generation was detected immediately after the photosensitization experiments by using a 2′,7′ dichlorodihydrofluorescein diacetate (H2DCFDA-AM) according to the manufacturer’s instruction. The culture medium of OECM-1 oral cancer cells that were exposed to 250 μg/mL nanocomposites (UCP@SiO2:MB-NRs and UCP@SiO2:MB-NRsFA) was first replaced with phosphate-buffered saline (PBS) containing 25 μM H2-DCFDA-AM and 0.1 μg/mL of DAPI that sufficiently covered the adhering cells. The cells were then subjected to photosensitization experiment by irradiation at 1.5 W/cm2 using CW 980 nm laser for 30 min. The fluorescent images of the cells stained with DCF (green emission at 520 nm) and DAPI (blue emission at 450 nm) were promptly captured by using the Leica TCS SP5 confocal microscope after excitations at 480 and 350 nm, respectively 2.5. Cell Viability Assay for PDT. Cell viability of the OECM-1 oral cancer cells treated with UCP@SiO2, UCP@SiO2-NP-FA, and UCP@SiO2-NR-FA for 24 h was assayed. OECM-1 cell lines were cultured in an incubator under 37 °C and 5% CO2 using the typical method. The cell medium used was RPMI-1640 medium mixed with 10% fetal bovine serum and penicillin/streptomycin L-glutamine. Approximately 2000/mL cells were loaded in each 96-well plate for 12 h for the 24 h treatment. Subsequently, 250 μg/mL nanocomposites were added to the plates, and the cells were incubated for 24 h. The experimental groups were divided according to irradiation with 980 nm laser for various durations. The control group was not irradiated. The resulting cell viability was calculated via an Alamar Blue assay using 560 EX nm/590 EM nm filter settings to determine fluorescence intensity. 32110

DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119

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ACS Applied Materials & Interfaces

Figure 2. (a, b) Transmission electron microscopy images of UCPs and UCP@SiO2 (c) Powder X-ray diffraction result of UCPs and UCP@SiO2, corresponding to the NaYF4 reference in the hexagonal phase. (d) Photoluminescence spectra of UCP@SiO2 and absorption spectra of UCP@ SiO2:MB. (1:1000, #9662, Cell Signaling, MA, USA). Anti-GAPDH was used as internal control (1:5,000, GTX100118, GeneTex, USA). Antisecondary antibodies were used to detect anticaspase-3 and anti-GAPDH (Sigma, USA). 2.9. Characterization. The size and morphology of samples were collected using a JEOL JEM-2100F electron microscope, and the crystalline phase of UCPs was characterized by 1.54 Å XRD pattern (Bruker D2 phaser diffractometer). The FT-IR spectra were obtained using a Varian FTIR-640 spectrometer. UC PL lifetimes were measured with a customized UV to mid-infrared steady-state and phosphorescence lifetime spectrometer (FSP920-C, Edinburgh Instrument) equipped with a digital oscilloscope (TDS3052B, Tektronix) and a tunable midband optical parametric oscillator pulse laser as the excitation source (410−2400 nm, 10 Hz, pulse width ∼5 ns, Vibrant 355II, OPOTEK).

before adding the ammonium solution to avoid the transformation to dimer structure, which would lead to the blue-shift of MB absorption.21 A fully covered silica shell with MB was synthesized after reaction with TEOS for 24 h. The thickness of the silica shell was adjusted by adding various amounts of TEOS, and the negative charge of TEOS improved the efficiency of drug loading because MB is positively charged. APTES was used to functionalize the amino group on the surface of the silica shell. FA and Au NRs were individually conjugated with UCP@SiO2-NH2 from the same active site for different reactions. FA reacted with the amino group on the surface through the coupling reaction of NHS/EDC, whereas the Au NRs were connected to the UCP@SiO2-NH2 via electrostatic force. Furthermore, Au NRs exchanged the protected ligand cetyltrimethylammonium bromide (CTAB) to O-carboxymethylchitosan to reduce the cytotoxicity from CTAB. The modification process was described in our previous study.21 The size and morphology of UCPs were directly characterized by transmission electron microscopy (TEM) after synthesis, and the results are shown in Figure 2a. The UCPs exhibited uniform spherical shapes with sizes of approximately 21.6 ± 0.9 nm. The TEM image in Figure 2b shows that majority of UCP@SiO2 nanocomposites were monodispersed and monocoated without aggregation, and the average thickness calculated from at least 50 particles was 4.5 ± 0.3 nm. The crystallinity of UCPs and UCP@SiO2 was measured by powder X-ray diffraction (XRD). The results shown in Figure 2c indicated that the UCPs and UCP@SiO2 possessed hexagonal phase, which could well match the standard hexagonal phase of NaYF4 in JCPDS No. 16-0334. The slightly

3. RESULTS AND DISCUSSION 3.1. Characterization of UCP@SiO2:MB-NRs. The fabrication of the UCP@SiO2:MB-NRs-FA nanocomposites is shown in Figure 1. Silica shell was coated with UCPs to embed the PS of MB and modify the FA and Au NRs. Silica shell was used because of its high biocompatibility and functionality. In addition, the PS of MB was embedded in the silica shell to improve photostability and prevent leakage during circulation. The UCPs, which are the core of the nanocomposites, were synthesized according to on our previous study,22 where we reported a high-temperature coprecipitation method; moreover, a silica shell was coated through reverse-phase microemulsion. Briefly, the surfactant of CO-520 was added in cyclohexane to construct a micelle and exchange the protected ligand of OA from UCPs under violent stirring. Afterward, MB was added 32111

DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119

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ACS Applied Materials & Interfaces

Figure 3. (a) Red emission of UC luminescence (UCL) spectra of UCP@SiO2:MB using various reactant volumes of MB (0, 50, 100, and 200 μL) during the coating of 4.5 nm thick silica shell (insert image shows the results of the full UCL spectra). (b) UCL spectra of UCP@SiO2:MB of silica shell with varying thickness (1.5, 4.5, 8.2, and 13.2 nm) using the same reactant volume of MB. The UCL emissions were detected from 400 to 700 nm under excitation using continuous wavelength (CW) 980 nm laser.

reactive space. The thickness of the silica shell was adjusted to 1.5, 4.5, 8.2, and 13.2 nm by changing the reactant volume of TEOS to 20, 40, 80, and 100 μL, respectively, under the same reaction time; the results are shown in Figure 3b. Unexpectedly, the declining trend of the red emission from UCL showed no dependence on the thickness of the silica shell. The highest reduction resulted from the middle thickness of 4.5 nm, followed by 8.2, 1.5, and 13.2 nm. This abnormal trend could be explained by the absorption spectra of UCP@SiO2:MB with different thicknesses of silica shell (Figure S2a). The absorption spectra of UCP@SiO2, similar to the UCPs, showed only one evident peak at approximately 980 nm. Thus, the characterized absorption from 500 to 700 nm could be attributed to the embedded MB in the silica shell. The different profiles of absorption spectra between each thickness referred to the change in the conformation of MB.25 Major absorption peak was observed at 655 nm for the 1.5 and 4.5 nm thicknesses, but the 13.2 nm thickness revealed a major absorption peak at 580 nm, and the 8.2 nm thickness was involved in the two conformation changes. MB absorption should be maximized at approximately 655 nm, not 580 nm, to achieve a highly efficient LRET between MB and UCP, because 655 nm well overlaps the red emission at 654 nm from the UCPs. We also tested the adding sequence for MB and ammonium solution (Figure S2b). MB should be added into the reaction before ammonium solution because the MB instantly reacts in the alkaline solution, thereby causing the dimerization of abundant MB and subsequent blue-shift absorption.26 Therefore, we inferred that the negatively charged TEOS would also cause some of the MBs to dimerize under high TEOS concentration. Furthermore, UCP@SiO2:MB was conjugated with Au NRs by enhancing the plasmonic field to further improve ROS production. The ratio of Au/Y and the UCL emission was determined to investigate the optimized condition of UCP@ SiO2:MB-NRs. Au NRs could enhance the UCL intensity in some cases via the plasmonic-field-enhanced emission or excitation rate in individual specific distances caused by the SPCE or LFE effect. The relationship between UCL intensity and ROS production is proportional.18−20 Au NRs could also enhance ROS production by increasing the light harvesting capability and absorption cross section in the system. This phenomenon is caused by the fact that organic MB usually present low absorption cross section of only ∼105 M−1cm−1 at 650 nm, and the common extinction coefficient of Au NRs is

enhanced background signal at approximately 30° was caused by the amorphous phase of silica shell. Moreover, we used a homemade photoluminescence machine to measure the UCL from NaYF4:Yb/Er NPs under excitation using laser with CW of 980 nm to confirm the UC radiative ability of NaYF4:Yb/Er NPs (Figure S1a). Figure S1c shows an evident absorption peak at approximately 980 nm, which could be attributed to the doping of Yb3+ in NaYF4. Strong multiemissions were observed at 408, 520, 540, and 654 nm. The transfer of energy diagram (Figure S1b) showed that Yb3+, as a sensitizer, exhibited a highabsorption cross section at 980 nm from the 2F7/2 → 2F5/2 transition. The emissions from Er3+ at 408, 520, 540, and 654 nm, which resulted from the absorption of few pumping photons from Yb3+, were denoted as 2H9/2 → 4I15/2, 2H11/2 → 4 I15/2, 4S3/2 → 4I15/2, and 4F9/2 → 4I15/2 transitions, respectively. Each emission could be used for distinct purposes based on the multiemission ability of UCPs. UCP@SiO2:MB showed high absorption peak at 655 nm, which well fit the irradiation from UCPs at 654 nm (4F9/2 → 4I15/2). This transition could facilitate the luminescence resonance energy transfer (LRET) between NaYF4:Yb/Er NPs and MB for ROS generation under laser irradiation with CW of 980 nm (Figure 2d). The efficacy of PDT was determined from three main factors, namely, oxygen, light, and PS. The typical mechanism of PDT is the excitation of the ground state of PS (singlet state) to the excited state of PS (triplet state) under irradiation. Subsequently, the excited state of PS would react with the surrounding oxygen to produce toxic ROS to further kill cancer cells.9,23 The amount of oxygen was determined in the inner tumor environment, which could not be easily changed in our therapeutic system; additionally, the light source would directly originate from the UCL. However, remarkable enhancement of the intensity of UCL is difficult because the UC process is based on 4f−4f electronic transitions, which are natural Laporte forbidden transitions.24 Therefore, the effective amount of PS was optimized to enhance the ROS generation initially. Figure 3a shows that increasing the reactant volume of MB (50, 100, 150, and 200 μL) during the coating of the silica shell remarkably reduced (13%, 51%, and 88%, respectively) the integrated area of the red emission at approximately 654 nm of UCL. The decreasing red emission directly corresponded to the loading amount of MB, which resulted in the LRET between MB and UCPs. Moreover, increasing the thickness of the silica shell required a large amount of MB because of the large 32112

DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119

Research Article

ACS Applied Materials & Interfaces

Figure 4. (a) Evaluation of reactive oxygen species (ROS) production of UCP@SiO2:MB and UCP@SiO2:MB-NRs on silica shell with varying thickness (1.5, 4.5, 8.2, and 13.2 nm) using the ROS marker 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) under irradiation at 1.5 W/ cm2 using CW 980 nm laser for 30 min. Phosphate-buffered saline (PBS) was used in the negative control group. (b) On−off examination of ROS production for UCP@SiO2, UCP@SiO2:MB, and UCP@SiO2:MB-NRs with 4.5 nm thick silica shell using ROS marker ABDA under irradiation of 1.5 W/cm2 using CW 980 nm laser for 60 min with 5 min intervals of on and off conditions. PBS was used in the negative control group. (c) ROS production of UCP@SiO2:MB, UCP@SiO2:MB-NPs, and UCP@SiO2:MB-NRs with 4.5 nm thick silica shell using ROS marker 1,3diphenylisobenzofuran under irradiation at 1.5 W/cm2 using CW 980 nm laser for 60 min. PBS and MB were used in the negative control groups. (d) Schematic mechanism of plasmon-enhanced ROS production for UCP@SiO2:MB-NRs.

∼109 M−1cm−1 at 650 nm.27−29 The ratio of Au/Y as base determination factor was determined by inductively coupled plasma mass spectrometry to verify the optimized amount of Au NRs for conjugation. We previously showed that the ratio of Au/Y in the UCP@SiO2:MB-NRs should be 0.2121 because this value displayed the highest enhancement of UCL intensity at 654 nm. In the present study, the different silica shells with 1.5, 4.5, 8.2, and 13.2 nm of UCP@SiO2:MB conjugated with Au NRs are shown in Figures S3a−S3e. The elemental confirmation of NaYF4:Yb/Er modified with Au NRs was determined through the energy-dispersive X-ray spectrometry (EDS; Figure S3f). 3.2. Evaluation of ROS Production by UCP@SiO2:MBNRs. The ROS production capability of UCP@SiO2:MB-NRs was verified in an aqueous solution using a previously identified optimized amount of MB and Au NRs. The efficacy of plasmon-enhanced ROS generation was also evaluated. The effective thickness of silica shell was first confirmed by using a ROS marker, namely, ABDA, and directly irradiating UCP@ SiO2:MB with varying silica shell thicknesses of 1.5, 4.5, 8.2, and 13.2 nm at 1.5 W/cm2 using laser with CW of 980 nm. The conformation of ABDA changed and caused the declining fluorescence signal at 407 nm under 380 nm excitation when ABDA reacted with the ROS (Figure S4a). Figure 4a shows that the tendency of ROS generation depended on the decreasing intensity of ABDA fluorescence signal with the

following order: 4.5 > 8.2 > 1.5 > 13.2 nm. This result is consistent with that of the absorption intensity of UCP@ SiO2:MB at 655 nm (Figure S2a). The highest loading amount of MB for the 4.5 nm thick silica shell could cause 30% reduction in fluorescence intensity at 407 nm. In addition, the 1.5 nm thickness caused an absorption intensity lower than that of 13.2 nm thickness at 655 nm, still revealing a slightly higher 10% ROS production than at 13.2 nm. This result is caused by the inverse proportionality between the efficiency of the energy transfer for the donor and acceptor and the distance of six square.30 Almost all thicknesses resulted in enhanced performance by 11.2%, 20.8%, 2.3%, and 0.2% for thickness of 1.5, 4.5, 8.2, and 13.2 nm, respectively. The variation was based on the effective distance between Au NRs and UCPs/MB. Three factors, namely, intensity of UCL, plasmonic field, and absorption cross section, were considered in the possible mechanism for ROS production enhancement. First, the intensity of UCL was enhanced using the sequence 1.5 > 4.5 > 8.2 > 13.2 nm by SPCE or LFE effect. As shown in Figure S5, the UCL intensity would be enhanced by 2.15- and 1.69-fold for 1.5 and 4.5 nm, but decreased by 0.97- and 0.83-fold for 8.2 and 13.2 nm by comparing the integrating areas of UCL spectra. The second factor was tested by transferring the excited electron (hot electron) to the PS from the Au NRs, which were excited with UCL.31 However, the MB of PS was embedded in the silica shell such that the excited electrons were difficult to 32113

DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119

Research Article

ACS Applied Materials & Interfaces

Figure 5. (a) Schematic working pathway of intracellular ROS sensor as 2′,7′ dichlorodihydrofluorescein diacetate. The ROS sensor should be treated before incubation with nanocomposites, and ROS production could be detected at fluorescence at 520 nm by 480 nm excitation from DCF. (b) Detection of intracellular ROS using UCP@SiO2:MB-NR and UCP@SiO2:MB-NRs-FA. The green fluorescent signal caused by ROS generation was detected using DCF, and blue fluorescent signal from the nucleus was detected using DAPI. Cellular medium was considered as the control.

Consequently, the UCL intensity was not considered to affect the ROS production under various thicknesses of silica shell under the same conditions. In addition, the decay time of UCL emission at 654 nm of UCP@SiO2, UCP@SiO2:MB, and UCP@SiO2:MB-NRs-FA with a thickness of 4.5 nm was investigated to study the energy transfer. As shown in Figure S7, the UCL decay time at 654 nm for UCP@SiO2:MB and UCP@SiO2:MB-NRs-FA was lower than that of UCP@SiO2 at 127, 95.4, and 195 μs. This obvious decrease revealed that the energy from UCL emission could transfer to MB or Au NRs through LRET. Additionally, the absolute quantum yield of nanocomposites was 0.4%, and no significant change was observed on UCPs. UCP@SiO2:MB-NR with 4.5 nm thickness of silica shell revealed the highest ROS production, which was almost twice that of UCP@SiO2:MB, because of the highest loading amount of MB and effective distance between Au NRs and UCPs/MB for LEF enhancement. UCP@SiO2:MB was modified with 4.5 nm of Au NPs with SPR band at the green region (approximately 520 nm) to verify the mechanism of plasmon enhancement and investigate the enhanced ROS production related to the good overlap between the absorption profile of MB and the SPR band of Au NRs. DPBF was used as the marker of ROS generation for absorption detection, and the principle of reaction was similar to that in ABDA. The conformation of DPBF changed by reacting with ROS, which led to the decreased absorption at 420 nm (Figure S4a). DPBF was used as ROS marker to determine the consistency of ROS detection using either fluorescence or absorption detection. Figure 4c shows that the decreasing tendency of UCP@ SiO2:MB-NRs was almost the same at approximately 60%

transfer to the PS by breaking through the silica shell insulator. For the third factor, the aspect ratio was changed according to the tunable SPR band of Au NRs to adjust the SPR peak and subsequently match the absorption of MB. This process caused the enlargement of the absorption cross section of MB through LEF, which is directly proportional to the distance.17,31 Therefore, the 4.5 nm thickness presented the highest enhancement because of its highest loading amount of MB and excellent effective distance for LEF operation. Furthermore, an “on−off” investigation of UCP@SiO2:MB and UCP@ SiO2:MB-NRs using ABDA was conducted to confirm the ROS production based on irradiation using CW 980 nm laser. Figure 4b shows the weakening intensity of fluorescence from ABDA for UCP@SiO2:MB and UCP@SiO2:MB-NRs under the on condition. Reduced levels of 60% and 35% were observed after irradiation for 60 min with 5 min intervals of on and off conditions. Compared with PBS at pH 7.4 and UCP@SiO2, which showed no change in fluorescence signal, the results indicated that the weakening fluorescence intensity directly corresponded to ROS generation. The UCL intensities of UCP@SiO2 with different thicknesses of silica shell were measured to evaluate the effect of variation in thickness. Figure S6a shows that the UCL intensity weakened for the fully covered silica shell coating but without obvious discrepancy under different thicknesses of 1.5−13.2 nm. Nevertheless, the weakening UCL intensity between each site at 408, 520, 540, and 654 nm varied (Figure S6b). This phenomenon probably indicated that shorter wavelength of emission need more pumped photon, and the precursor of silica shell, namely, TEOS, would quench the energy transfer from Yb3+ to Er3+.32 Thus, only the green emissions were primarily influenced. 32114

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Figure 6. (a) Photodynamic therapy (PDT) efficacy evaluated by cell viability assay for each group (UCP@SiO2-FA, UCP@SiO2:MB-FA, UCP@ SiO2:MB-NPs-FA, and UCP@SiO2:MB-NRs-FA). The groups were treated with OCEM-1 oral cancer for 12 h and examined with and without irradiation at 1.5 W/cm2 using CW 980 nm laser for 3 min. Cellular medium was considered the control group. (b) Time-dependent irradiation of PDT. OCEM-1 cells were treated with UCP@SiO2:MB-FA, UCP@SiO2:MB-NPs-FA, and UCP@SiO2:MB-NRs-FA for 12 h and examined with and without irradiation at 1.5 W/cm2 using CW 980 nm laser under different irradiation times.

soaking in medium for 1, 6, 12, 24, and 48 h through the DLS detection meant that nanocomposites might not cause any negative effect from aggregation. OECM-1 oral cancer cell was selected as the examination model because of the high expression of the FA receptor in this cell compared with other oral cancer cells.36 The selection of oral cancer was based on further exposure to the outside of body and easy treatment under laser exposure.37,38 In Figure 5a, the intracellular ROS marker acetate ester form of H2-DCFDA-AM, which is a membrane permanent molecule that passes through the cell membrane, was used to confirm whether the UCP@SiO2:MBNR nanocomposites could generate ROS intracellular production. Cellular esterases activated this ROS sensor to become nonfluorescent H2-DCFDA after H2-DCFDA-AM entered the cell. The nonfluorescent H2-DCFDA is ionic in nature and could be trapped inside the cell. This molecule would transform again into DCF when reacted with ROS, thereby revealing a strong green emission under 480 nm excitation. As shown in Figure 5b, the result of UCP@SiO2:MB-NRs for intracellular ROS study showed that the obvious green fluorescent emission could be detected compared with that treated with cellular medium by irradiating at 1.5 W/cm2 using CW 980 nm laser for 30 min. ROS production was improved after modifying FA on the surface (notable green fluorescent emission appeared around the nucleus). This phenomenon indicated that the intracellular uptake of nanocomposites could be increased through FA modification to enhance the production and further kill the cancer cells. 3.3. PDT Efficacy of UCP@SiO2:MB-NRs-FA in Vitro and in Vivo. The PDT capability of UCP@SiO2:MB-NRs-FA for the death of OCEM-1 oral cancer cell is shown in Figure 6a. The therapeutic efficacy of the different groups in vitro was tested by incubating individual samples with OECM-1 cell for 12 h. First, all UCP@SiO2-FA, UCP@SiO2:MB-FA, UCP@ SiO2:MB-NPs-FA, and UCP@SiO2:MB-NRs-FA caused no obvious cell apoptosis. The cell viability of all groups remained above 90%, which indicated the low cytotoxicity and high biocompatibility of the nanocomposites to the cell without 980 nm laser irradiation. Afterward, the original efficacy of PDT

under irradiation with CW 980 nm laser for 60 min. This result confirmed the absence of variation in measurement. The result for UCP@SiO2:MB-NPs revealed slight enhancement in ROS production of approximately 5% compared with that without any Au NM conjugation, but lower than that of UCP@ SiO2:MB-NRs by approximately 16%. This phenomenon indicated that the SPR band might be slightly red-shifted close to the absorption profile of MB by the coupling effect between Au NPs on the silica shell surface.33,34 In the present study, the UCP@SiO2:MB-NRs still resulted in good enhancement because the original extinction coefficient of Au NRs was higher than that of the Au NPs by approximately from 2-fold to 3-fold.29,35 Moreover, the SPR band of Au NRs revealed a good overlap with the absorption profile of MB (Figure S4b). The PBS and MB groups also exerted no change in the absorption intensity of DPBF, which indicated that PBS or MB could not produce ROS under irradiation with CW 980 nm laser. Thus, the mechanism of enhanced ROS production using UCP@ SiO2:MB-NRs with 4.5 nm thickness of silica shell could be verified using two strategies. First, enhanced red emission of UCL directly produces ROS through LRET from UCPs to MB because of the SPCE or LFE effect. Second, well-overlapped absorption profiles of MB and the SPR band of Au NRs would enlarge the absorption cross section of MB through the LFE effect (Figure 4d). The UCP@SiO2:MB-NR nanocomposites revealed their high potential for PDT based on the extracellular investigation of ROS generation. FA was grafted on the UCP@SiO2:MBNRs for intracellular studies to further improve the specific targeting ability to cancer cell. Whether FA was successfully modified on the UCP@SiO2:MB-NRs was confirmed; the FTIR spectra of UCP@SiO2:MB-NRs-FA showed stretching peaks at 1080 cm−1, which were assigned to Si−O bonding, and 1652 and 2920 cm−1, which were assigned to CO and C−H bonding; these peaks proved that silica shell and FA were modified around the UCPs (Figure S8a). In addition, the longterm stability of UCP@SiO2:MB-NRs-FA in the medium was examined before the in vitro study. As shown in Figure S8b, the size of UCP@SiO2:MB-NRs-FA without obvious change under 32115

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Figure 7. (a) Photograph of the setup for the PDT treatment in vivo test. (b) Relative tumor weight of UCP@SiO2:MB-NR-FA after the CW 980 nm laser treatment (insert image shows the photograph of tumor sizes of UCP@SiO2:MB-NR-FA after the CW 980 nm laser treatment). A significant reduction of tumor volume (50%) was observed in the UCP@SiO2:MB-NR-FA injection with CW 980 nm laser treatment group compared with control (*P < 0.05).

to CW 980 nm laser with beam expander under 200 mW/cm2 for 30 min with 5 min intervals of on and off conditions to prevent the damage caused by localized heat from the laser. The sample was injected on the third week when the tumor size was approximately 100 mm3, which might present angiogenesis and be similar to the actual condition in organism,39,40 and the results are shown in Figure S10b. Laser treatment showed that only the UCP@SiO2:MB-NRs-FA under laser irradiation could inhibit tumor growth during the fourth and fifth weeks, during which the tumors were smaller (approximately 200 mm3) compared with those of the other group. The weight of the mouse remained unchanged when PBS and UCP@SiO2:MBNRs-FA were injected with or without laser irradiation at the fifth week. This result indicated that the treatment exerted no adverse effect to the health of the mouse (Figure S10c). The mouse was sacrificed on the fifth week because the tumor overgrowth was remarkably large. The tumor weight and size with UCP@SiO2:MB-NRs-FA after laser treatment were reduced by almost 2-fold (Figure 7b), which showed the excellent PDT efficacy. The remarkable difference in therapeutic performance between the measured tumor size in the mice before sacrifice and actual tumor size after sacrifice could be attributed to the severe inflammatory phenomenon inside the tumor. After the cross section of the tumors was carefully examined and stained with H&E, six representative images of the tumor volume representing each treatment group were obtained. Our results showed the images from UCP@ SiO2 (−980 nm laser, Figure S10d, upper left panel), UCP@ SiO2 (+980 nm laser, Figure S10d, upper middle panel), and UCP@SiO2:MB-NRs-FA (−980 nm, Figure S10d, upper right panel) with all of the tumors with similar volumes after comparisons. However, the three representative images from UCP@SiO2:MB-NRs-FA after 980 nm laser treatment showed significant smaller tumor volumes (Figure S10d, lower panel) compared with those of the above control groups. Altogether, this evidence showed that UCP@SiO2:MB-NRs-FA injection and 980 nm laser irradiation significantly reduced the tumor sizes. Notably, treatment with UCP@SiO2:MB-NRs-FA on simulation of actual tumor condition could inhibit tumor growth, which confirmed the high efficacy and capability of UCP@SiO2:MB-NRs-FA for PDT in vivo. The major pathway of PDT shows the apoptosis process, which is highly related to the damage in the mitochondria resulting in a series of responses following an apoptotic pathway known as mitochondrion-mediated apoptosis (in-

without plasmon enhancement was obtained by comparison with the results from UCP@SiO2-FA and UCP@SiO2:MB-FA. UCP@SiO2:MB-FA caused a reduction of approximately 30% in cell viability under irradiation at 1.5 W/cm2 using CW 980 nm laser. UCP@SiO2:MB-NPs-FA and UCP@SiO2:MB-NRsFA revealed higher therapeutic efficacies than UCP@SiO2:MBFA (approximately 55% and 70%, respectively, vs 30% cell viability) when the efficiency of plasmon-enhanced ROS production in PDT was examined. The cause of cell death was verified given that Au NMs are also common NMs for PTT. In Figure S9, the temperature change of UCP@SiO2:MB and UCP@SiO2:MB-NRs-FA with Au/Y 0.21 under irradiation with CW 980 nm laser at 1.5 W/cm2 showed that temperature exhibited almost no obvious change; similar to H2O, this Au/Y condition was considerably low to generate sufficient heat for PTT. Therefore, we could confirm the cell death resulting from toxicity of ROS by PDT not PTT. The percentages of cells killed were 30%, 45%, and 70% for UCP@SiO2:MB-FA, UCP@ SiO2:MB-NPs-FA, and UCP@SiO2:MB-NRs-FA, respectively. These results were also consistent with the tendency of ROS production, thereby indicating that the dead cell could be attributed to the toxic ROS. The result of time-dependent irradiation of UCP@SiO2:MB-FA, UCP@SiO2:MB-NPs-FA, and UCP@SiO2:MB-NRs-FA also showed the increasing percentage of dead cells depending on laser irradiation time. UCP@SiO2:MB-NRs-FA showed the highest efficiency in PDT (almost 75% reduction in viability), which was higher than 20% of UCP@SiO2:MB-NPs-FA (Figure 6b). Hence, UCP@ SiO2:MB-NRs-FA was selected to evaluate the PDT performance in vivo. Further in vivo study was performed to investigate the effective operation on the organism on the basis of the high PDT efficacy of UCP@SiO2:MB-NRs-FA in vitro; UCP@ SiO2:MB-NRs-FA could produce abundant ROS in aqueous solution and intracellular condition, and cause obvious cell death through laser exposure. The two xenograft tumors were obtained by injecting the OECM-1 subcutaneously into the two sides of the backside to eliminate the deviation from each mouse (Figure S10a). UCP@SiO2 and UCP@SiO2:MB-NRsFA were selected as groups for comparison to verify the PDT efficiency. UCP@SiO2 and UCP@SiO2:MB-NRs-FA were injected intratumorally into the tumors on the same mouse, and the mouse was classified into with or without irradiation using CW 980 nm laser. The actual situation of PDT in vivo test is shown in Figure 7a. The individual tumors were exposed 32116

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Figure 8. (a) Mitochondrial membrane potential of UCP@SiO2:MB-FA and UCP@SiO2:MB-NR-FA stained by JC-1 and detected by confocal microscopy. The high-membrane potential resulted in the JC-1 aggregation in the membrane, as shown by the strong red fluorescent signal, and lowmembrane potential caused the monodispersed release of JC-1 to the cytosol, as shown by the strong green fluorescent signal. DAPI was used to mark the nucleus site. (b) Caspase 3 activities of the (1) control, (3) UCP@SiO2, and (5) UCP@SiO2:MB-FA without laser exposure. Rows 2, 4, and 6 show the Caspase 3 activities of the control, UCP@SiO2, and UCP@SiO2:MB-FA groups irradiated with CW 980 nm laser, respectively. (c) Schematic diagram of PDT treatment using UCP@SiO2:MB-NPs-FA and the cell apoptosis pathway.

trinsic pathway).41−43 Therefore, the pathway for cell apoptosis was determined by UCP@SiO2:MB-NRs-FA treatment with laser irradiation. Simple examination to verify the condition of mitochondria was conducted first. The ΔΨm is an early event in apoptosis. Thus, JC-1 staining was used to evaluate the alterations in ΔΨm. Healthy and normal mitochondrion membranes showed aggregation of JC-1 dye in the membrane (red emission). By contrast, unhealthy or damaged mitochondrion membrane exhibited the release of JC-1 dyes into the cytosol, which resulted in monodispersion and green fluorescence emission. Confocal microscopy was used to detect the intracellular fluorescent signal from JC-1 (Figure 8a). The UCP@SiO2:MB-FA and UCP@SiO2:MB-NR-FA revealed stronger green fluorescent emission and weaker red emission compared with the control group upon irradiation with CW 980 nm laser. These phenomena indicated that the mitochondria were damaged and might have been injured by toxic ROS from the nanocomposites. The quantitative value could also be calculated from the fluorescent image in Figure S11. The UCP@SiO2:MB-NRs and UCP@SiO2:MB-NPs-FA revealed weak red fluorescent signal as 7 and 9 units and strong green fluorescent signal as 4 and 16 units after laser treatment. The activity of caspase 3, which is an apoptosis marker, was tested to further determine whether the damage in the mitochondria induced the serious apoptosis pathway. Figure 8b shows that only UCP@SiO2:MB-NPs-FA revealed the high expression of the cleaved forms of caspase 3 (17 and 19 kDa), and 35 kDa of the pro-form caspase 3 disappeared. These phenomena referred to UCP@SiO2:MB-NPs-FA, which confirmed the induction of OCEM-1 cell death through the apoptosis pathway and could be attributed to the toxic ROS causing further damage to the mitochondria. The schematic of PDT treatment from UCP@SiO2:MB-NPs-FA is shown in Figure 8c. UCP@SiO2:MB-NPs-FA could produce a large

amount of toxic ROS through plasmon-enhanced PDT under irradiation with CW 980 nm laser. The ROS damaged the mitochondria and decreased the ΔΨm. These characteristics released the cytochrome-C to induce cell apoptosis by confirming the expression of caspase 3 activity.

4. CONCLUSIONS We demonstrated a plasmon-enhanced PDT strategy by using NaYF4:Yb/Er UCP nanocomposites. The nanocomposites were modified with Au NRs, and MB was embedded as PS in silica shell to improve the photostability and loading amount of MB. The UCL and absorption analysis of UCP@SiO2:MBNRs revealed that 4.5 nm was the optimized thickness of the silica shell for the loading amount of MB and effective distance for plasmon-enhanced ROS production. The performance could be improved with almost twice efficacy. This characteristic resulted in the highest ROS production in aqueous solutions and under intracellular conditions. This phenomenon could be attributed to the enhancement of the red emission of UCL by SPCE or LFE effect and the good overlapping of absorption profile of MB and SPR bands of Au NRs. As a result, the absorption cross section of MB was enlarged through LFE effect. Thus, UCP@SiO2:MB-NRs-FA provided highly promising multifunctional nanocomposites for oral cancer PDT on the basis of the enlargement capability for ROS generation in vitro and in vivo. The cell death pathway was also evaluated by JC-1 staining and caspase 3 activity. The highly toxic ROS damaged the mitochondrial membrane, which could be induced by the cell apoptosis pathway.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b07770. 32117

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Schematic of homemade UCL detector machine, UC energy transfer diagram, absorption spectra of UCP@ SiO2:MB, TEM images and EDS analysis of UCP@ SiO2:MB conjugated with Au NRs, schematic mechanism of ABDA and DPBF detect the ROS production, overlapping diagram of absorption spectra of MB, Au NPs, and Au NRs, UCL intensity of UCP@SiO2, schematic procedure of in vivo study, weight of mouse, weight of tumor, size of tumor, and calculation of the fluorescent intensity from the confocal image (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mai: [email protected]. *E-mail: [email protected]. ORCID

Michael Hsiao: 0000-0001-8529-9213 Ru-Shi Liu: 0000-0002-1291-9052 Author Contributions

All authors contributed to the manuscript. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the Ministry of Science and Technology of Taiwan (Contract Nos. MOST 104-2113-M002-012-MY3, MOST 104-0210-01-09-02, and MOST 1050210-01-13-01) and Academia Sinica (Contract No. AS-103TP-A06) for the financial support for this research. We are also grateful to Ms. C.Y. Chien of the Precious Instrument Center (National Taiwan University) for her assistance in the TEM experiments.

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REFERENCES

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DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119

Research Article

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DOI: 10.1021/acsami.6b07770 ACS Appl. Mater. Interfaces 2016, 8, 32108−32119