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Article Cite This: J. Phys. Chem. C 2018, 122, 2402−2412

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Single 808 nm Laser Treatment Comprising Photothermal and Photodynamic Therapies by Using Gold Nanorods Hybrid Upconversion Particles Ming-Hsien Chan,†,‡,¶ Shao-Pou Chen,‡,§,¶ Chieh-Wei Chen,† Yung-Chieh Chan,∥ Ren Jie Lin,⊥ Din Ping Tsai,⊥,# Michael Hsiao,*,⊥,○ Ren-Jei Chung,*,§ Xueyuan Chen,‡ and Ru-Shi Liu*,†,∥,#,∇ †

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan CAS Key Laboratory of Design and Assembly of Functional Nanostructures, and Fujian Key Laboratory of Nano-materials, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China § Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei 106, Taiwan ∥ Genomics Research Center, Academia Sinica, Taipei 115, Taiwan ⊥ Department of Physics, National Taiwan University, Taipei 106, Taiwan # Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan ○ Department of Biochemistry College of Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan ∇ Department of Mechanical Engineering and Graduate, Institute of Manufacturing Technology, National Taipei University of Technology, Taipei 106, Taiwan

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S Supporting Information *

ABSTRACT: Light therapy has become the subject of research on cancer treatment because of its selectivity, low invasive damage, and side effects. Photothermal therapy (PTT) and photodynamic therapy (PDT) are prevalent treatments used to induce cancer cell apoptosis by generating heat and reactive oxygen species (ROS). In this study, mesoporous silica shellcoated gold nanorods (AuNR@mS) are synthesized by seed crystal growth method. AuNR@mS are assembled into nanocomposites through electrostatic adsorption with lanthanidedoped upconversion nanoparticles (UCNP). When controlling the aspect ratio of gold nanorods (AuNRs), the surface plasmon resonance peaks of the short-axis and the long-axis match the maximum absorption cross section at 520 and 660 nm of the fluorescence light released by the UCNPs. The converted fluorescence stimulates AuNRs to generate heat through energy transfer. ROS production is induced by loading the photosensitizer Merocyanine 540 (MC540) in the mesoporous silica layer and is further enhanced through the surface plasma resonance effect of the AuNRs. This novel nanoplatform combines PTT and PDT in a single 808 nm near-infrared synergistic light therapy.

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INTRODUCTION

with traditional surgery and chemotherapy, which have many side effects and restrictions, light therapy is more suitable for oral cancer therapy because of its less invasiveness and fewer side effects.5 Light treatment is mainly divided into photothermal therapy (PTT)6 and photodynamic therapy (PDT).7 Since the unique growth mechanism of malignant tumor cells, a concentration gradient may make the drug molecules hard to enter when the tumor is formed. It means that only using the PTT or PDT is not enough to completely kill the cells. The combination of these light therapies is a breakthrough in the

With the aging population, malignant tumors have become the most critical battlefield in the field of biomedical engineering. Cancer cells have a special growth mechanism because of their abnormal hyperplasia and neovascularization, which accelerate the transport of nutrients; in this regard, a single treatment method cannot lead to tumor necrosis. Hence, different therapy methods should be combined to avoid cancer cell recurrence.1,2 The occurrence of oral cancers is high in Asian countries, especially in South Asia. The Asian cultural practice of betel nut chewing, along with the use of tobacco or alcohol, may raise the risk that predisposes a person to cancer of the oral cavity.3 Oral cancer is one of the most common cancers and can be treated with common surgery or chemotherapy.4 However, compared © 2018 American Chemical Society

Received: November 7, 2017 Revised: December 16, 2017 Published: January 5, 2018 2402

DOI: 10.1021/acs.jpcc.7b10976 J. Phys. Chem. C 2018, 122, 2402−2412

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The Journal of Physical Chemistry C current bottleneck of cancer treatment. Sun et al. used dendrimers of gold nanorods (AuNRs) as the core and grafted photosensitizers with upconversion nanoparticles (UCNPs) to form core−satellite-type composites for diagnosis and multitreatment of mouse tumors.8 In previous studies, UCNPmediated PDT was conducted by 980 nm laser excitation, and the AuNR-mediated PTT was induced by 808 nm laser.9−12 These studies proved that double-therapy methods can be a competitive cancer treatment. In the current study, the excitation light of the photosensitizer is generally located in the ultraviolet and visible (UV−vis) wavelength bands; the expansion of light therapy in the biomedical field remains challenging due to the limitations of short wavelength and penetration depth. As such, the sensitizers Yb3+ and Nd3+, and the activator Er3+ were doped to produce UCNPs, which excite visible light through high penetration of 808 nm near-infrared (NIR) light.13 The 808 nm laser can effectively prevent the absorption of hemoglobin and water molecules, and the upconverted energy can be efficiently transferred to the photosensitizer for light therapy.14,15 Using UCNPs combined with AuNRs, Zhan et al. regulated the aspect ratio of AuNRs to individually overlap their short-axis and long-axis absorption peaks with the excitation light source of the UCNPs.16 In this paper, the authors ensure that the dopant ions were all affected by the electromagnetic field of the surface plasma resonance (SPR) to enhance the effect of the PTT.17 Furthermore, a hybrid plasmonic upconversion nanostructure (AuNR@ UCNPs) exhibits strongly enhanced upconverted fluorescence (up to 20 folds).18 The combination of PTT and PDT of the photosensitizer should be driven by two lasers. For instance, one of the NIR light laser drives PTT, and the other visible laser light drives PDT. This treatment process is cumbersome, and visible light is an unsuitable light source for light therapy.19 In this study, an AuNR with an aspect ratio of 2.86 was used as photosensitizer for PTT. The specific ratio of the AuNRs was chosen to match the 541 and 654 nm fluorescence released by Er3+-doped UCNPs, with the short- and long-axis absorption peaks, respectively. This process induces the gold materials to produce heat by the SPR effect.20−22 The photosensitizer, Merocyanine 540 (MC540), was carried in mesoporous silica layer can simultaneously absorb the 541 nm fluorescence released by the upconversion material and produce reactive oxygen species (ROS) for PDT. MC540 can accumulate for a long time in tumor cells, thereby enhancing the effect of PDT by the plasma resonance generated by the AuNRs. This nanocomposite is a new generation of theranostic platform, which can be applied to combine PTT with PDT under singlechannel laser irradiation (Scheme 1).

Scheme 1. Schematic Illustrating That Upconversion Nanoparticles Conjugating with Photosensitizer-Loaded Gold Nanorods Are a New Generation of a Theranostic Platform, Which Can Be Applied To Combine Photothermal Therapy with Photodynamic Therapy Under Single-Channel Laser Irradiation

through “Tetra Pak method,” and the use of CTAB as soft template can generate a silica layer.25 After removing CTAB by acid solution, mesoporous silica-coated AuNRs (AuNR@mS) were synthesized (Figure S1c).26 As shown in Figure S1, parts a and b, AuNRs and AuNR@mS exhibit excellent dispersibility in aqueous solutions. In the spectral test, the absorption intensity of AuNR@mS at 520 nm slightly increased, and the long-axis absorption peak shifted about 10 nm due to the change in the surface dielectric constant (Figure S1d). The nitrogen adsorption and desorption isotherm of AuNR@mS is type IV, confirming the pore structure of AuNR@mS (Figure S1e). On the basis of the BET-BJH theory, the pore size of the mesoporous silica layer is 2.3 nm. The specific surface area of AuNR@mS is 288 m2/g, and the pore volume is 0.457 m3/g (Figure S1f). The synthesized silica layer belongs to the mesoporous type and can be used for loading photosensitizers or drugs. Second, coprecipitation was employed to synthesize UCNPs. NaYF4:Yb/Er is the core of the UCNP, the second shell is coated with the NaYF4:Yb/Nd layer, and the third shell is the NaYF4 layer (Figure S2, parts a, b, and c).27 However, hydrophobic carbon chain ligands, which are hydrophilic and dispersed in the aqueous environment, must be removed to use UCNPs in the biomedical field. The absorption of previous UCNPs with long CH2 chains at 1457, 1557, 2853, and 2925 cm−1 was tested by Fourier-transform infrared spectroscopy (FTIR) analysis. After the removal of surface oleic acid by weak acid treatment, the characteristic peaks of oleic acid molecules disappeared (Figure S2d). UCNPs were maintained the βNaYF 4 hexagonal phase (JCPDS 16−0334) after any modification (Figure S2e), and its crystal structure of is shown in Figure S2f. Lastly, nanocomposites were assembled by electrostatic attraction force. The AuNR@mS was combined with UCNP by the interaction between the negative silica layer and the positive poly L-lysine (PLL) layer (Figure 1a).28 The morphology and particle size of AuNR@mS and UCNP-PLL were identified through the transmission electron microscopy (TEM) analysis. The results are shown in Figure 1, parts b and c. The nanocomposite, namely, AuNR@mS-MC-UCNP, was synthesized through the above assembly step (Figure 1d). With the barrier of the mesoporous silica layer, a space between the AuNR and UCNP prevents the fluorescence of UCNP being quenched by AuNR. The element distribution of AuNR@mSMC-UCNP was analyzed by X-ray energy dispersive spectroscopy (EDS). The EDS data show that Na and F, which are light elements, are uniformly located in the UCNP. In contrast to

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RESULTS AND DISCUSSION In this research, AuNRs were controlled to match the position of the emission peaks of the UCNPs. Moreover, the photosensitizer, Merocyanine 540 (MC540), as carried in mesoporous silica layer was also match the position of the emission peaks of the UCNPs and could produce reactive oxygen species (ROS).23 This novel nanosystem dispersed in saline solution was driven by a single-channel 808 nm laser to achieve both PTT and PDT. Furthermore, in vitro and in vivo experiments were conducted to confirm the therapeutic effects of the nanocomposites on the biological environment. First, the toxic surfactant cetyltrimethylammonium bromide (CTAB) was found on the surface of AuNRs due to the seed growth process.24 Tetraethoxysilan was applied as silicon source 2403

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Figure 1. (a) Synthesis of AuNR@mS-MC-UCNP. (b) TEM image of AuNR@mS. (c) Focused TEM image showing a layer of polymer-modified PLL on UCNP. (d) TEM and (e) HAADF-STEM-EDS mapping images of AuNR@mS-UCNP (scale bar is 40 nm).

about 22 mV on the surface after binding with PLL. Finally, the AuNR@mS-MC-UCNP has a surface potential of −2 mV. The loading amount of MC540 was measured based on the characteristic absorption peak via the supernatant by centrifugation. Loading concentration was calculated by the calibration line. As shown in Figure S3d, the loaded MC540 amount of AuNR@mS-MC-UCNP is 14.3 mg/g, with a drug loading rate of 43%. The size distribution of particles is determined with dynamic light scattering (DLS) spectrum. (Figure S4) We transfer the AuNR@mS and AuNR@mS-MCUCNP into simulated body fluid. The hydraulic radius of the AuNR@mS and AuNR@mS-MC-UCNP are 99.52 and 148.9 nm, respectively. The optimal penetration of NIR in the biological tissues was found at 808 nm because excitation at this wavelength can prevent the absorption of water and hemoglobin. In this study, the upconverted mechanism of UCNP is shown in Figure S5a. The Nd3+ ions doped in the second shell absorb 808 nm NIR laser and pass through the 4I9/2 → 4F5/2 transition to the excited state; the ions quickly relax to the 4F3/2 order because of the unstable order of 4F5/2, then through the 4F3/2 → 2F5/2 of the cross-relaxation phenomenon to transfer energy to Yb3+. After energy transfer into Er3+, it possesses the characteristics of the emission spectrum at the core layer (Figure S5b). The result show that the UCNP The characteristic emission peaks of Er3+ from high energy to low energy are 2H9/2 → 4I15/2 (blue light at 410 nm), 2H11/2 → 4I15/2, 2S3/2 → 4I15/2 (green light at 522 and

these light elements, the middle signal of Au completely matches with the distribution of nanorods (Figure 1e). UCNP exhibits significant signal in the region because of the doping of the core and shell with Y and Yb. Er is only doped in the core layer, and Nd is found at the part of the shell layer. FTIR analysis was conducted to detect changes in the functional groups on the surface and confirm the assembly process of AuNR@mS and UCNP-PLL (Figure S3, parts a and b). The surface of AuNRs, synthesized by seed growth method is protected by the surfactant CTAB. The signals at 2853 and 2925 cm−1 represent the hydrophobic carbon chain CH2 of CTAB molecules. After the silica layer coated the AuNRs, the spectrum shows the characteristic peak of Si−O−Si at 1080 cm−1. The FTIR pattern reveals the characteristic peak of the siloxy functional group, but no CH2 signal was observed after reflux with weak acid. This result indicates that the soft template CTAB was removed and successfully formed into the AuNR@mS. The PLL polymer was modified on UCNP to cover the positively charged ions. The FTIR spectra of UCNP show the surface functional groups at 1000 (CO bond); 1130, 1250, and 1440 (CO bond); and 1650 cm−1 (NCO bond), corresponding to PLL. Surface electrical properties were detected by ζ potential (Figure S3c). AuNR possesses surface potential of about 26 mV because its surface carries positively charged cations. The surface potential of AuNR@mS is about −18.7 mV because of the cladding of the silica layer. UCNP, which is almost no charge on the surface, carries a potential of 2404

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Figure 2. (a) UV−vis absorption of AuNR@mS/MC540 and photoluminescence spectra of UCNP. (b) Emission spectra with UCNP, UCNP-PLL, and AuNR@mS-UCNP determined using 808 nm laser. (c) Simulative time-averaged energy density in mesoporous silica shell. (d) Quality and distribution of electric field and time-averaged energy density on AuNR@mS-UCNP under irradiation of individual UCL emission at 410, 520, and 660 nm.

540 nm), and 4F9/2 → 4I15/2 (red light at 662 nm).29 The upconverted fluorescence can transfer to AuNR and the photosensitizer MC540. The fluorescence of the radiation wavelength should accurately overlap the absorption peaks of AuNR and MC540 to obtain the maximum conversion effect of heat and photodynamics (Figure 2a). UCNP radiation fluorescence wavelength not only overlaps with the shortand long-axis absorption peaks of AuNR@mS at 520 and 660 nm, respectively, but also covers the absorption of MC540 with the largest cross-sectional area. After the composition of the nanocomposite AuNR@mS-MC-UCNP, the fluorescence wavelengths of 522, 540, and 662 nm are significantly reduced. The descending ratios of green and red wavelengths are 52.5% and 70.6%, respectively. This decline can be attributed to the absorption by AuNR@mS and MC540 (Figure 2b). We measured the luminous lifetime of UCNP for identification of efficiency of energy transfer, and observed the change of fluorescence lifetime after AuNR@mS-MC modified (AuNR@ mS-MC-UCNP). The luminous lifetime of 540 nm band is observed the change from 0.11 to 0.06 ms and 660 nm band is observed the change from 0.56 to 0.25 ms under the excitation of 808 nm laser (Figure S5c and d). As a result, the decrease of lifetime demonstrated that the upconverted bands are greatly absorbed at 660 nm light and slightly absorbed at 540 nm because the absorption of AuNR and MC540, respectively. To evaluate the energy distribution, an inversed dipole and an electric field with time were applied in the x-direction of UCNP (E⃗ = E⇀0eiωt, E⇀0 = −3εP⇀0), and observed the electric dipole moment in the electromagnetic radiation properties.30 Theory of the electric dipole moment radiation is in accordance

with the following theoretical formula of electromagnetic radiation by eq eq 1). Therefore, the direction of the electromagnetic wave of UCNP radiation is perpendicular to the electric dipole moment (Figure S6; hence, the direction of radiation in the xz plane is the z direction, and the yz plane of the radiation is in the form of spherical waves.31 E=

⎫ ⎛1 1 ⎧ ω2 iω ⎞ ⎨ 2 (r ̂ × p) × r ̂ + ⎜ 3 − 2 ⎟[3r(̂ r ·̂ p) − p]⎬ ⎝r 4πε0 ⎩ c r ⎭ cr ⎠ eiωt / ce−iωt

(1)

After confirming the simulation, the relationship between the energy distribution of UCNP and AuNR@mS-MC was determined. To evaluate the emission of UCNP, we chose the wavelengths of 410, 520, and 660 nm, respectively. The fluorescence intensity of UCNP is the same benchmark at the three wavelengths. This study also investigated if the surface resonance effect of AuNR can increase the energy density at the silica layer. After the conversion of the electric field to energy density, the energy density is highest at 660 nm wavelength, followed by 520 nm, and the lowest at 410 nm (Figure 2d). The results are attributed to matching of the fluorescence wavelength of UCNP with the absorption wavelength of AuNR (Figure 2c). When the fluorescence wavelength is consistent with the peaks of SPR, the energy transfers and accumulates in AuNR. The energy density of the mesoporous silica layer increases due to the SPR effect, and the photosensitizer MC540 loaded in the layer is in the high-density energy state. The energy state can make the MC540 set in high-photon-density state and enhance the ROS production. To investigate the PTT 2405

DOI: 10.1021/acs.jpcc.7b10976 J. Phys. Chem. C 2018, 122, 2402−2412

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Figure 3. PTT effect evaluation: (a) temperature IR images of UCNP, AuNR@mS, AuNR@mS-UCNP, and AuNR@mS-MC-UCNP (1 mg/mL) irradiated under 808 nm laser (1.5 W/cm2) for 20 min; (b) time-dependent increase in the temperature of H2O, UCNP, AuNR@mS, AuNR@mSUCNP, and AuNR@mS-MC-UCNP (1 mg/mL) under 808 nm (1.5 W/cm2) irradiation for 10 min. PDT effect evaluation; (c) absorbance changes in ABDA treated with AuNR@mS-MC-UCNP under 808 nm light excitation; (d) photodegradation rate of ABDA in the presence of AuNR@mSMC, AuNR@mS-UCNP, UCNP-MC, and AuNR@mS-MC-UCNP (1 mg/mL) under 808 nm light (1.5 W/cm2) excitation for 60 min.

the absorption peaks of ABDA significantly decline. Compared with the control group, because the UCNP-MC can directly transfer the upconverted energy to MC540, the ABDA absorption decays quickly before 0−40 min. However, the AuNR@mS-MC-UCNP group shows the most decreased ABDA absorption under prolonged irradiation (Figure 3d). It proves that the SPR effect enhanced the energy transfer process. The AuNR@mS-MC and AuNR@mS-UCNP groups are unable to receive the energy of the 808 nm laser and the nonloaded photosensitizer without ROS production. Compared with these groups, the AuNR@mS-MC-UCNP group is loaded with MC540 into the mesoporous silica layer, and the SPR effect of the AuNR results in the high optical density state of MC540. The production of ROS could decrease the absorption intensity of ABDA light by about 40%. According to this process, we can expect that when the 808 nm NIR laser is used to excite the UCNPs, the emitted wavelength at 540 nm can transfer energy to MC540 to achieve PDT, and the emitted wavelength at 660 nm can transfer energy to AuNR to achieve PTT. Moreover, the SPR effect of the AuNR can further enhance those phototreatments. For the application of the synthesized nanocomposites in the biological field, the safety of materials should be assessed. In this study, in vitro experiments were performed using CAL 27 oral cancer cell lines for a series of tests. The nanocomposites were diluted 1/3 of the highest concentration of 250 μg/mL to form concentration gradients (250, 81, 27, 9, and 3 μg/mL), mixed with CAL 27 cells, and incubated for 48 h. After labeling the cells with Alamar Blue cell dyes, cell viability was evaluated through fluorescence analysis. As shown in Figure 4a, AuNR@

effect between UCNP and AuNR, we irradiated UCNP, AuNR@mS, AuNR@mS-UCNP, and AuNR@mS-MC-UCNP with 1.5 W at 808 nm NIR for 10 min. There is no significant temperature rise because of the 808 nm light does not overlap with the absorption spectrum of AuNR; however, AuNR can efficiently absorb the emission of 808 nm NIR-driven UCNP at 662 nm bands. The thermal image indicates that the AuNR@ mS-MC-UCNP (1 mg/mL) exhibits excellent light and heat transfer properties; thus, the temperature increases to more than 42 °C (Figure 3a). The temperature of the solution was recorded every 20 s, and a temperature rise curve was drawn (Figure 3b). The NIR laser at 808 nm avoids the maximum absorption range of water molecules; after 10 min of irradiation with pure water and individual UCNP solution, the temperature did not increase. However, with or without the loading of the photosensitizer MC540, the combination of UCNP and AuNR can be heated up the solution to 44 °C for 10 min to obtain outstanding light heating characteristic. ROS, such as singlet oxygen 1O2 and other free radicals, are cytotoxic to cells. We used 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) as fluorescent reagent to detect ROS produced by [email protected] ABDA reacts with ROS present in the solution and is transferred by an irreversible oxidation route of ABDA, resulting in decreased absorption coefficient. The absorption of ABDA was detected by UV−vis light absorption spectrometer at 359, 378, and 399 nm. The absorption peaks of ABDA were recorded in the AuNR@mS-MC-UCNP solution under 808 nm laser irradiation for every 10 min. ROS production is shown in Figure 3c. When the measured environment contains numerous singlet oxygen molecules, 2406

DOI: 10.1021/acs.jpcc.7b10976 J. Phys. Chem. C 2018, 122, 2402−2412

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Figure 4. (a) Cell viability after being treated with AuNR@mS, AuNR@mS-MC, AuNR@mS-UCNP, and AuNR@mS-MC-UCNP. (b) Under 808 nm laser irradiation for 5 min (1.5W/cm2). (c) TUNEL assay of CAL 27 cells immediately after being treated with 250 μg/mL AuNR@mS, AuNR@ mS-MC, AuNR@mS-UCNP, and AuNR@mS-MC-UCNP under 808 nm laser irradiation for 20 min (scale bar is 25 μm).

effect than PTT or PDT group alone. Apoptosis was assessed by TUNEL method (Figure 4c).33 DCFH2-DA can immediate entry to the cell and convert into DCFH by intracellular esterase. DCFH then reacts with intracellular ROS and is oxidized into DCF with the fluorescence emission characteristic. DCF can be excited by 488 nm to shine the green fluorescence emission at 533 nm (Figure 5c). Such fluorescent differences, we used the flow cytometry to confirm this result again and to indicate the presence of ROS in the cells (Figure 5a and b).34 After 10 min of laser irradiation, UCNP-MC and AuNR@mS-MC-UCNP emit green fluorescent light. Moreover, the AuNR@mS-MC-UCNP group could yield improved PDT effect. The results confirm that AuNR@mS-MC-UCNP could induce cancer cell apoptosis by PTT and PDT, resulting in improved outcomes of the light treatment. For in vivo experiments, we used the mouse as an animal model to evaluate the mechanism of phototherapy in cancer treatment. All of the mice were reared for 6 weeks beginning with the implantation of the tumor. In parts a and b of Figure 6, the fluorescence imaging of UCNP and AuNR@mS-MC-UCNP was performed with noninvasion in vivo imaging system (IVIS). We chose nude mice to prevent unnecessary hair shielding. As with the previous conditions, the up-converted fluorescence was excited by 808 nm NIR (500 mW) and received green fluorescence (520 nm) emitted by Er3+. The energy power has been

mS, AuNR@mS-MC, UCNP-MC, and Au@mS-MC-UCNP were maintained at more than 80% after coculture with the cells (3−250 μg/mL). The results show that all groups have no significant toxicity to the CAL 27 cell line. In cell phagocytosis, CAL 27 cells were incubated with 250 μg/mL AuNR@mS, AuNR@mS-MC, UCNP-MC, and Au@mS-MC-UCNP aqueous solutions for 12 h. Cell uptake was observed by confocal microscopy with nuclear staining (4′,6-diamidino-2-phenylindole, DAPI). DAPI showed the location of the nucleus, and UCNP was stimulated by 808 nm laser to emit 540 nm green fluorescence (Figure S7). In the confinement image of each group of nanomaterials and CAL 27 cells, greenest fluorescence overlapped or encircled at the cytoplasmic position. We then selected DCFH2-DA reagent as ROS probe to detect the intracellular presence of free radicals. We used an Alamar Blue assay to detect the cytotoxicity of the material and irradiated the sample with a 808 nm laser (1.5W/cm2) for 5 min to confirm the light treatment results of the nanocomposites. More than 90% survival rate is maintained in the AuNR@mS-MC group (Figure 4b). The survival rate of the CAL 27 cell line showed no significant toxicity. AuNR@ mS-UCNP and UCNP-MC were also driven by 808 nm light source and showed toxicity after incubation at high concentrations. However, AuNR@mS-MC-UCNP composites in the combined PTT and PDT demonstrate improved killing 2407

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Figure 5. Detection of intracellular ROS by using 250 μg/mL AuNR@mS-MC, AuNR@mS-UCNP, UCNP-MC, and AuNR@mS-MC-UCNP. All groups were treated with DCF as ROS probe. After irradiation with 808 nm laser for 20 min (1.5W/cm2), flow cytometry analysis shows the intensity of green fluorescence of (a) UCNP-MC and (b) AuNR@mS-MC-UCNP. (c) The fluorescent signal was detected using LSCM, the green fluorescent signal from the DCF dye, and the blue fluorescent signal from the nucleus was detected using DAPI (scale bar: 25 μm).

decreased to avoid the laser heat may hurt the skin of mice. Moreover, organs were evaluated by IVIS. All mouse organs did not show up-converted fluorescence, except for the tumor injected with AuNR@mS-MC-UCNP (Figure 6c). All organs were found to be under normal situation because despite the entry of the nanocomposites in the cells, NIR light treats the tumor, thereby preventing damage to the other organs. The phototherapy effect was also assessed by a white mouse model. We injected CAL 27 oral cancer cells in the left and right buttocks of the mouse, and the cells were maintained for 3 weeks to grow the tumor to around 125 mm3. All animals were categorized into three groups: AuNR@mS-UCNP for PTT, UCNP-MC for PDT, and AuNR@mS-MC-UCNP for PTT + PDT. In comparison with PTT and PDT alone, the PTT + PDT group shows the optimal therapy effect on reducing the tumor tissues (Figure 6, parts d and e). As a result, simply using the 808 nm laser to treat tumor tissues cannot make the size become smaller and/or lighter. Figure 6d shows that, the tumor weight has slightly decreased after treatment with PDT and PTT, respectively. Moreover, the PTT + PDT group shows the

best results for reducing the tumor weight. Figure 6e shows a photograph of the tumor tissue consolidating the results in Figure 6d. According to the in vivo results, multifaceted phototherapy (PTT + PDT) can significantly kill cancer cells after 2 weeks of treatment.

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EXPERIMENTAL SECTION Synthesis of Gold Nanorods. Gold nanorods (AuNRs) were synthesized by a modified seed-mediated growth method. Seed solution was prepared by adding NaBH4 (0.01M; 0.6 mL) to the mixed solution of HAuCl4 solution (0.1 M; 25 μL) and CTAB solution (0.2 M; 5 mL) under vigorous stirring for 5 min. The brown seed solution formed was stored at 30 °C for 1 h to hydrolyze the unreacted NaBH4. The growth solution was prepared by dissolving sodium salicylate (0.8 g) in the CTAB solution (0.1 M; 250 mL) and adding AgNO3 solution (4 mM; 6 mL). The mixture was kept at 30 °C for 15 min and added with HAuCl4 solution (1 mM; 250 mL). The solution was kept undisturbed for 15 min and added with fresh ascorbic acid solution (0.064 M; 1 mL) and preprepared seed solution (0.8 2408

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Figure 6. Animal model fluorescence imaging with 100 μL of the (a) UCNP and (b) AuNR@mS-MC-UCNP in suspended solution (10 mg/mL). (c) Organs fluorescent evaluation by IVIS system. (d) In vivo PTT (AuNR@mS-UCNP), PDT (UCNP-MC), and PTT + PDT (AuNR@mS-MCUCNP) evaluated the weight change of tumor by irradiation 808 nm NIR for 30 min (250 mW/cm2). (e) Tumor tissues were obtained to ensure the therapy effect of PTT, PDT, and PTT + PDT (w/o, without and w/, with the laser irradiation).

mL). The solution was incubated at 30 °C overnight to grow AuNRs. The nanorods were purified by centrifugation at 10,000 rpm for 30 min, condensed, and redispersed in distilled water. Silica Shell Coated on the Surface of AuNRs. TEOS (30 μL) was added to the AuNR solution (10 mL) in alkaline environment and incubated for 2 days to form mesoporous silica shell-coated AuNRs (AuNR@mS). The solution was refluxed with HCl at 60 °C for 6 h. The AuNR@mS was purified by centrifugation at 10,000 rpm for 20 min and redispersed in distilled water. Merocyanine 540 Loading. The photosensitizer Merocyanine 540 (MC540) was loaded in the mesopores of the silica shell. MC540 solution (6 mM, 10 μL) was added to the AuNR@mS solution, and the samples were rotated for 24 h. After MC540 loading, the solution was purified by centrifugation at 10,000 rpm for 20 min and redispersed in distilled water. Synthesis of NaYF4:Yb3+/Er3+ Nanoparticles. NaYF4:Yb,Er nanoparticles were synthesized by modified hightemperature coprecipitation method. The solution of Y(CH3CO2)3·H2O (0.2 M; 3.2 mL), Yb(CH3CO2)3·H2O (0.2 M; 0.72 mL), and Er(CH3CO2)3·H2O (0.2 M; 0.08 mL) were mixed in a 50 mL two-neck round-bottom flask with OA (6 mL) and 1-octadecene (14 mL). The solution was heated to 150 °C under nitrogen protection for 30 min and cooled to 50 °C. The solution was then added with the MeOH solution of NH4F (1 M; 2 mL) and NaOH (0.4 M; 7.9 mL) and stirred for 30 min. The reaction mixture was heated at 100 °C for 30 min to remove methanol. The mixture was then heated to 290 °C and kept for 2 h before cooling to room temperature. The asprepared nanoparticles were precipitated by adding absolute ethanol, collected by centrifugation at 7600 rpm for 6 min, and washed with ethanol for several times. The upconversion nanoparticles (UCNPs) were stored in cyclohexane for the next step.

Synthesis of NaYF4:Yb3+/Er3+@NaYF4:Yb3+/Nd3+ Nanoparticles. NaYF4:Yb3+/Er3+@NaYF4:Yb3+/Nd3+ NPs were synthesized using similar method, but Er3+ was replaced with Nd3+. Briefly, the solution of Y(CH3CO2)3·H2O (0.2 M; 3.4 mL), Yb(CH3 CO 2) 3 ·H 2O (0.2 M; 1.7 mL), and Nd(CH3CO2)3·H2O (0.2 M; 1.7 mL) was mixed in a 50 mL two-neck round-bottom flask with OA (6 mL) and 1octadecene (14 mL). The solution was heated to 150 °C under nitrogen protection for 30 min and cooled to 50 °C. The solution was then added with the preprepared NaYF4:Yb3+/Er3+ and MeOH solution of NH4F (1 M; 2 mL) and NaOH (0.4 M; 7.9 mL) and stirred for 30 min. The reaction mixture was heated at 100 °C for 30 min to remove methanol, then heated to 290 °C and kept for 2 h before cooling to room temperature. The as-prepared nanoparticles were precipitated by adding absolute ethanol, collected by centrifugation at 7600 rpm for 6 min, and washed with ethanol for several times. The UCNPs were stored in cyclohexane for the next step. Synthesis of NaYF 4 :Yb 3+ /Er 3+ @NaYF 4 :Yb 3+ /Nd 3+ @ NaYF4 NPs (UCNPs). The third shell was coated on the surface of UCNPs to stabilize NaYF4:Yb3+/Er3+@NaYF4:Yb3+/ Nd3+ NPs. The process was the same as described above, but the lanthanide solutions were replaced with Y(CH3CO2)3·H2O (0.2 M; 4 mL). The mixture was added with the as-prepared NaYF4:Yb3+/Er3+@NaYF4:Yb3+/Nd3+ NPs when the nanoparticles were formed. Synthesis of AuNR@mS−UCNP. The nanocomposites were assembled by electrostatic adsorption. The surface of UCNPs was winded with poly L-lysine hydrobromide (PLL, Mw = 1000 to 5000) to obtain positive charge. The modified UCNP solution was mixed with AuNR@mS−MC solution for 1 h to form AuNR@mS−UCNP nanocomposites. 808 nm NIR Laser-Driven Photodynamic Experiment. ABDA was used to react with 1O2 to detect ROS in the aqueous solution. AuNR@mS−MC, AuNR@mS−UCNP, UCNP−MC, and AuNR@mS−MC−UCNP nanocomposites (1 mg/mL) 2409

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The Journal of Physical Chemistry C were added to 1 mL of water containing 10 μM ABDA dye. The solution was placed in cuvette and stirred. The solution was then irradiated with 1.5 W/cm2 808 nm laser for 60 min. ROS production was confirmed by detecting the absorption of ABDA at 378 nm. The absorbance spectra of the mixture after NIR-laser irradiation were recorded by Fluoromax-3 (Horiba, Japan). After NIR-laser irradiation, the absorption spectra of the mixture were recorded by UV-1700 spectrophotometer (Shimadzu, Japan). Simulative Analysis. In this study, numerical simulation of the electromagnetic module in COMSOL 3.5a was carried out, which was a set of numerical calculation based on finite element method (FEM). As the structure of the design of the size of the nanolevel, which caused the surface plasma resonance mainly occurs in the bands of visible and near-infrared. The evaluation of radio frequency worked by Maxwell Calculation and Analysis of Maxwell Equation. Because of the free electrons that can move freely inside the metal, these free electrons are dominated by the applied behavior of the applied electromagnetic field. Therefore, this study uses the Drude-Lorentz model to simulate the structure of the gold nanorods. Since the nanocomposites are dispersed in the water environment, the refractive index of the mesoporous silica layer and the upconversion nanoparticles were set to 1.33, 1.8 and 1.46, respectively. The energy density of the nanocomposites is calculated by applying an electric field to according to the following formula eq 2 and eq 3. ⎛ 1 1 1 ⎞ E ⃗=⎜ x̂ + ŷ + z⎟̂ ⎝ 3 3 3 ⎠

Uave =

d(ωμμ0 ) 2 ⎤ 1 ⎡ d(ωεε0) 2 ⎢ |E | + |H | ⎥ 4 ⎣ dω dω ⎦

1% triple antibiotic reagent. Both cell lines were incubated in 5% CO2 incubator at 37 °C. Briefly, every well containing 2000 cells was inoculated in 96-well plate for 12 h. Different diluted concentrations of the AuNR@mS−MC−UCNP solution (3, 9, 27, 81, and 250 μg/mL) were treated for another 24 h. Under the NIR treatment condition, all cells were exposed to irradiation under 808 nm NIR laser (1.5 W/cm2) for a short time. After 48 h of incubation, Alamar Blue assay was conducted, and measurements were performed using SpectraMax M2 (Molecular Devices, California, USA) at excitation/ emission of 560/590 nm. Detection of in Vitro PDT Effect. Intracellular ROS generation was detected immediately after the photosensitization experiments by using H2-DCFDA reagent. The DMEM culture medium containing CAL 27 oral cancer cells exposed to 250 μg/mL nanocomposites (AuNR@mS−MC−UCNP) was replaced with PBS containing 25 μM H2−DCFDA and 0.1 μg/ mL DAPI to completely cover the adhering cells. The cells were then subjected to photosensitization experiment by irradiation at 1.5 W/cm2 by using 808 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 immediately captured by the Leica TCS SP5 confocal microscope after excitation at 480 and 350 nm, respectively. Animal Tumor Model Imaging. An InGaAs CCD camera was used to obtain NIR image (Princeton Instruments) and ensure the biodistribution of the AuNR@mS−MC−UCNP conjugates in vivo. The 808 nm excitation light was provided by a fiber-coupled diode laser (250 mW, Hi-Tech Optoelectronics Co., Ltd.) with 4.5 cm laser extender counter. The energy power has been decreased to avoid the unnecessary heat made from 808 nm laser. The emitted light that passed through an 830 nm long-pass filter (Semrock) was taken as the signal. Briefly, 100 μL of the AuNR@mS−MC−UCNP suspended solution (10 mg/mL) was administered by intratumoral injection for the tumor imaging of the tumor-bearing mice. Detection of in Vivo PDT Effect and Animal Tumor Model. Animal experiments were approved by the Institutional Animal Care and Utilization Committees of Academia Sinica (IACUC No. 16-05-957). CAL 27 cells (5 × 106 cells/100 μL) were injected subcutaneously on the left and right thigh of the mouse. AuNR@mS−MC−UCNP (100 mg) was intratumorally injected into the tumor when tumors had grown to 125 mm3. Animal fluorescence was evaluated under irradiation with 250 mW/cm2 of 808 nm NIR-laser and performed by noninvasion in vivo imaging system (IVIS) after 12 h of incubation for 30 min under 5 min intervals of cooling down and irradiation. All tumors were acquired after 1 week. The tumor sections were fixed on formalin and embedded on paraffin.

(2)

(3)

ε0 = vacuum permittivity, ε = relative permittivity of the material, μ0 = vacuum permeability, and μ = relative permeability. Cellular Uptake and Localization of AuNR@mS−MC− UCNP. Oral adenosquamous and oral epidermoid carcinoma cell lines (CAL 27 cell lines, respectively) were selected as in vitro models. The two cell lines were seeded in six-well plates at a density of 20,000 cells/mL and incubated overnight. The plates were then incubated with AuNR@mS, UCNP, and AuNR@mS−MC−UCNP at 37 °C for 12 h. Confocal and TEM images were obtained to confirm the localization of the particles. For confocal imaging, the cells were incubated and washed with PBS to remove nanocomposites that were not internalized. The cells were fixed on a glass chip by adding 1 mL of 4% paraformaldehyde per well for 5 min. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min to label the cells. The control and experimental groups were observed by confocal microscopy. The nucleus image was excited at 408 nm, and emission was detected from 450 to 500 nm. The AuNR@mS−MC−UCNPs were excited at 808 nm, and the emission was detected from 450 to 500 nm. For TEM imaging, the cells were stripped by trypsin, centrifuged, fixed with glutaraldehyde (2.5%), embedded in pure resin, cut into ultrathin sections, and stained by osmic acid. Finally, the uptake phenomena of AuNR@mS−MC−UCNPs were examined by TEM imaging with a field emission gun working at 80 kV. In Vitro Cytotoxicity of AuNR@mS−MC−UCNP with/ without 808 nm NIR-Laser Irradiation. For cytotoxicity assay, CAL 27 cell lines were separately grown in DMEM and RPMI-1640 medium containing 10% fetal bovine serum and

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CONCLUSION In summary, UCNPs were excited by the near-infrared light at 808 nm by controlling the surface plasma position of AuNR to release the fluorescence at 520 and 660 nm. The SPR peaks of the AuNRs was controlled by the addition of silver nitrate and sodium salicylate to absorb the fluorescence of the UCNPs under 808 nm near-infrared irradiation. Construction of singlechannel 808 nm laser excitation, while the formation of photothermal and photodynamic therapy of nanocomposite materials. The photosensitizer MC540 was loaded through the mesoporous silica layer coating to absorb the unconverted fluorescence in a high optical density state, resulting in cytotoxic ROS. By the SPR effect of the gold nanorods, the 2410

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space of silica layer has an amplitude increase to improve the effect of photodynamic therapy. We recommend the construction of single-channel 808 nm laser excitation and the formation of PTT and PDT nanoplatforms.

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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/acs.jpcc.7b10976. Experimental methods and figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

Din Ping Tsai: 0000-0002-0883-9906 Michael Hsiao: 0000-0001-8529-9213 Xueyuan Chen: 0000-0003-0493-839X Ru-Shi Liu: 0000-0002-1291-9052 Author Contributions ¶

These authors contributed equally to this work.

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 No. MOST 104-2113-M-002012-MY3) for financially supporting this research. This research was also supported by Academia Sinica and Ministry of Science and Technology (Contract No. MOST 106-0210-01-15-02, MOST 107-0210-01-19-01). We are grateful to C. Y. Chien of the Precious Instrument Center for the assistance in TEM experiments and to Ms. L. W. Lo and S. C. Hsu of the Academia Sinica for the help with the confocal microscopy. The authors acknowledge financial support from Ministry of Science and Technology, Taiwan (Grant No. MOST-106-2745-M-002003-ASP). They are also grateful to National Center for Theoretical Sciences, NEMS Research Center of National Taiwan University, National Center for High-Performance Computing, Taiwan, and Research Center for Applied Sciences, Academia Sinica, Taiwan, for their support. X.C. acknowledges the support from the NSFC (Nos. U1305244, 21325104) and the CAS/SAFEA International Partnership Program for Creative Research Teams.

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