Single 808 nm Laser Treatment Comprising Photothermal and

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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 J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b10976 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

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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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 shell-coated gold nanorods (AuNR@mS) are synthesized by seed crystal growth method. AuNR@mS are assembled into nanocomposites

through

electrostatic

adsorption

with

lanthanide-doped

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.

INTRODUCTION


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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. It’s because the cultural practices of Asian such as the betel nut chewing and the addiction of tobacco or alcohol may raise the risk that predisposes 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 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 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 multi-treatment of mouse tumors.8 In previous studies, UCNP-mediated 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 double3 ACS Paragon Plus Environment


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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 longaxis 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 4 ACS Paragon Plus Environment

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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 single-channel laser irradiation (Scheme 1).

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 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 S1a 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 5 ACS Paragon Plus Environment


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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). Based on 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, co-precipitation 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 S2a, 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, and 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 β-NaYF4 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 1b 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@mS-MC-UCNP was analyzed by X-ray 6 ACS Paragon Plus Environment

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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 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 S3a 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 Zeta 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 about 22 mV on the surface after binding with PLL. Finally, the AuNR@mS-MCUCNP 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



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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-MC-UCNP into simulated body fluid. The hydraulic radius of the AuNR@mS and AuNR@mS-MC-UCNP are 99.52 nm 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 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 short- and 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-MCUCNP, 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 8 ACS Paragon Plus Environment

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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 0.11ms to 0.06 ms and 660 nm band is observed the change from 0.56 ms 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 ( = , = −3 ), 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 equation (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 z direction, and the yz plane of the radiation is in the form of spherical waves.31

=

̂ × × ̂ + " # − $ %3̂ ̂ ∙ − '( / *

(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



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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 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-MCUCNP (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 10 ACS Paragon Plus Environment

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[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, 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 mins. 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 non-loaded 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 photo-treatments.



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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@mS, AuNR@mS-MC, UCNP-MC, and Au@mS-MC-UCNP were maintained at more than 80% after co-culture 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’,6diamidino-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. 12 ACS Paragon Plus Environment

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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 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 mouse as animal model to evaluate the mechanism of phototherapy in cancer treatment. All of the mice were reared for six weeks beginning the implantation of tumor. In Figure 6a and b, the fluorescence imaging of UCNP and AuNR@mSMC-UCNP was performed with non-invasion 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 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 13 ACS Paragon Plus Environment


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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 6d 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 show 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.

■ EXPERIMENTAL SECTION Synthesis of Gold Nanorods. Gold nanorods (AuNRs) were synthesized by modified seedmediated growth method. Seed solution was prepared by adding NaBH4 (0.01M; 0.6 mL) to the 14 ACS Paragon Plus Environment

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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 pre-prepared seed solution (0.8 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 was 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 high-temperature co-precipitation 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 15 ACS Paragon Plus Environment


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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 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 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(CH3CO2)3·H2O (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 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 pre-prepared 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 NaYF4:Yb3+/Er3+@NaYF4:Yb3+/Nd3+@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


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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) 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 nano-level, 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 Drude17 ACS Paragon Plus Environment


Page 18 of 34

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). E = " ./ + √-

7 7

U345 = 6

√-

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Single 808 nm Laser Treatment Comprising Photothermal and

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