Journal of Nanomaterials
Volume 2016 (2016), Article ID 1082746, 10 pages
http://dx.doi.org/10.1155/2016/1082746

Research Article

Gold Nanorods-Based Theranostics for Simultaneous Fluorescence/Two-Photon Luminescence Imaging and Synergistic Phototherapies

Shan Fang,¹ Chunxiao Li,¹ Jing Lin,² Haigang Zhu,¹ Daxiang Cui,³ Yunsheng Xu,¹ and Zhiming Li¹

¹Department of Dermatology and Venereology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China
²Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, Department of Biomedical Engineering, School of Medicine, Shenzhen University, Shenzhen 518060, China
³Department of Bio-Nano Science and Engineering, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, China

Received 13 November 2015; Accepted 11 January 2016

Academic Editor: Jibin Song

Copyright © 2016 Shan Fang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Gold nanorods (GNRs) have shown great potential applications in cancer theranostics due to the unique phenomenon of surface plasmon resonance, which leads to strong electric fields on the surface and consequently enhances the absorption and scattering in the near-infrared (NIR) region. Indocyanine green (ICG), an amphipathic dye, is not only an excellent NIR imaging agent but also an ideal light absorber for laser-mediated photodynamic and photothermal therapy. In this study, in order to integrate the merits of GNRs and ICG in biomedical applications, we developed ICG conjugated silica-coated GNRs (GNR@SiO₂-ICG) for cancer imaging and phototherapy. The covalent coupling strategy reduces the probability of leakage/desorption during the delivery. The as-prepared GNR@SiO₂-ICG could serve as efficient probes to simultaneously enhance fluorescence (FL) imaging and two-photon luminescence (TPL) imaging. In vitro experiments indicated that A375 cells could be killed through synergistic phototherapies effect of GNRs and ICG using single wavelength continuous-wave laser irradiation. Our results indicated that the synthesized GNR@SiO₂-ICG are effective for simultaneously enhancing FL/TPL imaging and synergistic phototherapies.

1. Introduction

Photothermal therapy (PTT), a therapeutic method to convert photon energy into heat to destroy target cells, has been increasingly investigated as a minimally invasive alternative to oncological treatments [1, 2]. To achieve the desired treatment effect, the following requirements are essential: (1) tumor localization: various imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), fluorescence imaging, and photoacoustic (PA) imaging, have been implemented to define location and size of the tumor. (2) Photothermal conversion agents (PTCAs) choice. An ideal PTCA should possess little or no biotoxicity, good biocompatibility, effective accumulation at tumor sites, high photothermal conversion efficiency, and so on [3, 4]. (3) Laser modes regulation. For example, nanosecond pulsed laser is highly selective and localized damage controllable but is low heating efficiency. Comparatively, continuous-wave (CW) laser can effectively accumulate heat content but is time consuming [5]. For biological applications, the energy output intensity and time should be as low as possible to avoid damage to the surrounding healthy tissues of tumor. (4) Temperature monitoring. Ultrasound imaging [6] and thermal imaging [7] have been widely used to real-timely and noninvasively monitor the temperature variation in tumor region during the process of PTT.

Because of unique surface plasmon resonance (SPR), plasmonic (noble metal) nanoparticles, especially gold nanoparticles, are able to quickly and effectively convert absorbed photon energy into heat in the picosecond time domain [8, 9], which make them excellent candidates for hyperthermia cancer treatment. Among differently shaped gold nanoparticles, gold nanorods (GNRs) have been studied most extensively due to facile synthesis and surface modification, excellent biocompatibility and biodegradability, superior tunable optical properties and photostability, long blood circulation time, and good cellular affinity [10]. Previous studies indicated that GNRs exhibit higher absorption efficiency than nanocages or nanoshells with the SPR at the same wavelength [10], thus making it possible to achieve the same therapeutic effect at a lower laser intensity. It is worth mentioning that, through varying the aspect ratios (length/width), the longitudinal surface plasmon resonance (LSPR) peak of GNRs can be made tunable in near-infrared (NIR) region where light penetration is optimal due to the minimal absorption by tissue chromophores and water [11]. Furthermore, the high scattering cross sections of GNRs render them good contrast agents for dark field microscopy imaging and two-photon photoluminescence (TPL) imaging in cancer cell imaging [12]. Thus, GNRs are believed to be more promising in biomedical applications.

Indocyanine green (ICG), an amphipathic tricarbocyanine dye, is the only NIR organic dye approved by the US Food and Drug Administration (FDA) for human medical imaging and diagnosis in clinical applications [13]. It exhibits strong absorption band at around 800 nm and the most intense fluorescence at around 820 nm. ICG molecules can absorb and transfer energy from light to molecular oxygen, leading to the generation of reactive oxygen species (ROS) including singlet oxygen (SO). In addition, it is able to absorb the energy of corresponding wavelength and easily reach the excited state and then release the energy to the surrounding tissue when returning to their ground state, thereby elevating the temperature in the process [14, 15]. Therefore, ICG is also an ideal NIR light absorber for laser-mediated photodynamic and photothermal therapy. Unfortunately, several physicochemical characteristics have badly restricted biological applications of ICG, such as short circulation half-life, high protein binding capacity, low quantum yield, easiness of fluorescence quenching, poor stability in aqueous solution and blood plasma, complex relationship between fluorescence intensity and concentration, and limited availability of functional groups for conjugation [12]. Recently, numerous polymeric nanoparticles (NPs) and inorganic NPs are introduced to encapsulate or adsorb ICG molecules, thus improving stability or prolonging circulation time. However, some inevitable defects are possible to exist, including complex synthesis process, large particle size [16], fluorescence resonance energy transfer (FRET) induced fluorescence quenching [17], and rackety incorporation.

In this work, we developed a novel ICG-containing nanostructure GNR@SiO₂-ICG for dual-functional imaging and phototherapies therapy. Firstly, gold nanorods (GNRs), as bright contrast agents for two-photon luminescence (TPL) imaging and photothermal therapy (PTT), were synthesized using a seed-mediated growth method. The LSPR band was tuned into near-infrared region. Secondly, in order to reduce toxicity, provide basic for further modification, and enhance stability as well as biocompatibility, silica was coated on the surface of gold nanorods by hydrolysis and condensation with tetraethoxysilane (TEOS). Finally, indocyanine green N-succinimidyl ester (ICG-NHS) possesses succinimidyl ester active groups which provide an efficient and convenient way to selectively and covalently link ICG dyes to primary amines (R-NH₂) on GNR@SiO₂-NH₂, thus reducing the probability of leakage/desorption during the delivery process of nanocomposites to tumor [18]. Our in vitro experiments results showed that internalized GNR@SiO₂-ICG could be used for TPL and fluorescence imaging. The synergistic phototherapies effect of combination of GNRs and ICG was evaluated in vitro, simultaneously.

2. Materials and Methods

2.1. Materials

Chloroauric acid (HAuCl₄·4H₂O), sodium borohydride (NaBH₄), silver nitrate (AgNO₃), sulfuric acid (H₂SO₄), L-ascorbic acid (AA), ammonia (NH₃·H₂O), and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (3-Aminopropyl) triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Cetyl trimethyl ammonium bromide (CTAB) and anhydrous dimethyl sulfoxide (DMSO) were obtained from Yeasen Corporation (Shanghai, China). Cell culture products and reagent, unless mentioned otherwise, were purchased from Gibco (Tulsa, OK, USA). All the above chemicals were used without any further purification. Ultrapure water (18.2 MΩ·cm, Millipore Co., USA) was used in all the preparations.

2.2. Synthesis of GNRs and GNR@SiO₂

GNRs were synthesized according to the well-developed seed-mediated growth method [19]. Briefly, 7.5 mL 0.1 M CTAB was first mixed with 102.9 μL 24.3 mM HAuCl₄ solution and 1.8 mL ultrapure water. Then, 600 μL 0.01 M ice-cold NaBH₄ was quickly added into the mixture and vigorously stirred for 2 min at 30°C. The solution was protected from light and shaken at 30°C until the formation of Au seeds. After 2 h, 800 μL 10 mM AgNO₃ was added into the mixture of 100 mL 0.1 M CTAB, 2.06 mL 24.3 mM HAuCl₄, and 2 mL 0.5 M H₂SO₄, followed by 800 μL 0.1 M ascorbic acid that was added under vigorous stirring for 2 min. Finally, in order to initiate the growth of the GNRs, 240 μL of seed solution was immediately injected to the above mixture solution at 30°C under gentle stirring for 30 s. The transparent solution was kept at 30°C overnight to obtain GNRs.

Mesoporous silica coating on GNRs was performed according to modified stöber method [20]. 20 mL as-prepared GNRs was centrifugated (11000 rpm, 15 min, once) to remove excess CTAB. The residue was redispersed in 20 mL ultrapure water. Then, 15 μL ammonia (ca. 28 wt%) was added under vigorous stirring to adjust pH to ca. 10. After that, 30 μL 10% TEOS in ethanol was slowly injected four times at 20 min intervals under gentle stirring, and the reaction mixture was allowed to react for 24 h at 30°C.

2.3. Preparation of GNR@SiO₂-NH₂ and GNR@SiO₂-ICG

The obtained GNR@SiO₂ were washed by centrifugation (6800 rpm, 15 min) with ultrapure water and ethanol for twice, respectively, to remove the residual CTAB. Then, the nanoparticles were dispersed in 4 mL ethanol and then completely distributed by ultrasonication, followed by 4 mL APTES that were added directly into the above GNR@SiO₂ product and allowed to react under refluxing at 80°C for 4 h. The resultant was washed with ethanol and ultrapure water for several times and then redispersed in PBS solution (pH 8.5) to form a homogeneous dispersion and for further use.

100 μL 10 mg/mL ICG-NHS (Intrace Medical SA Co., Ltd., Lausanne, Switzerland) was added into the above as-prepared purified GNR@SiO₂-NH₂ samples under ultrasonication for 2 h at room temperature and protected from light. The terminal NHS groups of the functional ICG derivative were specifically reacted with the primary amino groups on the surface of GNR@SiO₂. The product was washed and purified by centrifugation until the supernatant became colorless and cannot detect its characteristic ultraviolet absorption peak. The covalent binding amount of ICG-NHS to the GNR@SiO₂-NH₂ was analyzed using a UV-Vis spectrophotometer at 800 nm.

2.4. Characterization

The particle size and morphological structure were performed by transmission electron microscopy (TEM) with JEM-2100F (JEOL, Japan), operating at an acceleration voltage of 200 kV. UV-Vis spectra were measured with a Varian Cary 50 spectrophotometer (Varian Inc., Palo Alto, CA, USA). Fluorescence spectra were acquired on a Hitachi FL-4600 spectrofluorometer. Zeta potential of the samples was completed using a NICOMP 380 ZLS Zeta Potential/Particle Sizer (PSS Nicomp, Santa Barbara, CA, USA).

2.5. Cell Culture and Cytotoxicity Assessment

Human melanoma cell line A375 was obtained from the cell store of Chinese Academy of Science. All cells were routinely cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM, HyClone) containing 10% (vol/vol) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% CO₂. All experiments were performed on cells at the logarithmic growth phase.

A375 cells were seeded in 96-well plates with a density of 5 × 10³ cells/well and incubated overnight. After being rinsed with PBS (pH 7.4, 1 mM), the cells were exposed to GNRs, GNR@SiO₂, GNR@SiO₂-NH₂, and GNR@SiO₂-ICG with various concentrations (0–80 μg Au/mL) for 24 h under the same culture conditions. Then, 10 μL cell counting kit-8 (CCK-8, Yeasen Corporation Shanghai, China) was added into each well. After being incubated for additional 3 h, the optical density for each well was read by a microplate reader (Thermo, Varioskan Flash) at a dominant wavelength of 450 nm with a reference wavelength at 630 nm.

2.6. Bio-TEM and Confocal Laser Scanning Microscopy (CLSM) Image

A375 cells were seeded into 3.5 cm plates at a density of 1.0 × 10⁵ cells and allowed to attach for 24 h. Medium was replaced by fresh medium containing 10 μg Au/mL GNR@SiO₂-ICG and then continually incubated for 24 h. The untreated cells served as control group. All cells were washed carefully with PBS for several times and harvested by centrifugation (2000 rpm, 2 min). All samples were fixed in 2.5% (w/w) glutaraldehyde at 4°C overnight, respectively. TEM specimens were embedded and prepared according to standard procedures, and then the morphology of GNRs in A375 cells was observed by biotransmission electron microscopy (Bio-TEM, JEOL JEM-1230).

The intracellular two-photon luminescence (TPL) that came from GNRs and two-photon excitation fluorescence (TPEF) that came from ICG could be imaged by Leica TCS SP8 confocal laser scanning microscopy. Briefly, A375 cells (5 × 10⁴ cells/well) were seeded on glass cover slips placed in 24-well culture plates and allowed to adhere for 24 h. Medium was replaced by fresh medium containing 10 μg Au/mL GNR@SiO₂-ICG. After 24 h of incubation, cells were washed carefully with PBS for several times and then fixed with 2.5% (w/w) glutaraldehyde for 20 min. Cells cultured under normal conditions were used as control group. For TPL imaging, the excitation wavelength was 800 nm and the emission wavelength was 520 nm. For fluorescence imaging, the excitation wavelength was 650 nm and the emission wavelength was 780 nm.

2.7. Photothermal Therapy (PTT) Effect In Vitro

To investigate the photothermal effect of GNR@SiO₂-ICG induced by NIR laser irradiation, 400 μL GNR@SiO₂-ICG (1000 μg Au/mL) aqueous solutions were exposed to 808 nm continuous-wave diode laser (LWIRL 808–5000 mW Beijing Laserwave Optoelectronics Technology Co., Ltd.) with an output power of 1.86 W/cm² for 8 min. GNR@SiO₂-NH₂ and PBS under the same experiment conditions served as control group. The temperature elevation was monitored every minute by a thermocouple (187 True RMS Multimeter, Fluke).

A375 cells (6 × 10³ cells/well) were seeded in 96-well plates for 24 h. Medium was replaced by fresh medium containing 10 μg Au/mL GNR@SiO₂-NH₂ or GNR@SiO₂-ICG and subsequently cultured for 24 h. The photothermal treatment was performed using an 808 nm laser at an output power of 1.86 W/cm² for 0, 1, 2, or 3 min, respectively. All cells were incubated for another 12 h. The cell viability was measured using CCK-8 assay.

For Calcein AM (Dojindo Laboratories)/propidium iodide (PI, Sigma-Aldrich Co., LLC.) costaining experiment, A375 cells were seeded in 24-well plates with a density of 5 × 10⁴ and attached for 24 h. Cells were incubated with 10 μg Au/mL GNR@SiO₂-NH₂ or GNR@SiO₂-ICG for 24 h and then irradiated by an 808 nm laser at a power density of 1.86 W/cm² for 3 min. After being incubated for another 6 h, all cells were washed with PBS carefully and stained with PBS solution containing Calcein-AM (2.5 μg/mL) and PI (2.6 μg/mL) for 20 min and then imaged by a confocal fluorescence microscope (Nikon Eclipse 80i).

3. Results and Discussion

3.1. Synthesis and Characterization of GNR@SiO₂-ICG

GNRs were first prepared according to the seed-mediated method. The cetyltrimethylammonium bromide (CTAB) formed a bilayer around the GNRs and served as a template for formation of a silica layer. The aspect ratios of GNRs could be controlled by changing the concentration of reductive agent silver nitrate and L-ascorbic acid. Well-defined GNR@SiO₂ was obtained by using an improved stöber method, and the thickness of silica layer was determined by final amount of TEOS. The functionalized GNR@SiO₂ was prepared via cocondensation method by reacting TEOS with APTES in ethanol. GNR@SiO₂-ICG could be effectively synthesized by covalent binding of succinimidyl ester active groups of ICG-NHS with GNR@SiO₂-NH₂ under alkaline conditions.

Figure 1 showed the TEM images; the GNRs core had an average length of ca. 51.2 nm, width of ca. 16.9 nm, and aspect ratio of ca. 3.1. The mesoporous silica shell on the surface of GNRs was quite homogeneous and uniform with a thickness of ca. 20.3 nm, which prevents direct contact between ICG molecules and the GNRs core, and thus guaranteeing weak or inefficient fluorescence resonance energy transfer (FRET). Because of the low electron density of APTES and ICG-NHS, there were no apparent changes in the shape and size of GNR@SiO₂ after modificating amine group and conjugating ICG molecules.

Figure 1: Representatively TEM images of GNRs, GNR@SiO₂, GNR@SiO₂-NH₂, and GNR@SiO₂-ICG. Scale bars, 50 nm.

The UV-Vis spectra of nanoparticles dispersed in aqueous solution were shown in Figure 2(a). As expected, GNRs show a weak transverse plasmon band at around 512 nm and a strong longitudinal plasmon band at around 800 nm. After silica-coated GNRs, the longitudinal plasmon band exhibits a small redshift (~23 nm) because the silica shell increases the local refractive index of the medium surrounding the GNRs [21]. The longitudinal plasmon band of GNR@SiO₂-NH₂ showed negligible shift but obvious damping contrasted to GNR@SiO₂, indicating that the amine groups have been successfully modified on surface of mesoporous silica shells. A further redshift accompanying shape change was observed upon ICG conjugation. Furthermore, the absorption band of GNR@SiO₂-ICG caused obvious redshift and enhancement compared with free ICG-NHS at the same concentration (Figure 2(b)). These results confirm that ICG has been successfully coupled with GNR@SiO₂-NH₂. ICG-NHS standard curve of absorption value versus concentration was plotted and the coupling rate of ICG was estimated to be 37.2 wt%.

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Figure 2: (a) UV-Vis absorption spectra of GNRs (black), GNR@SiO₂ (blue), GNR@SiO₂-NH₂ (green), and GNR@SiO₂-ICG (red). (b) UV-Vis absorption spectra of GNR@SiO₂-ICG (red) and ICG-NHS (black). (c) Fluorescence spectra of GNR@SiO₂-ICG and free ICG-NHS at the same concentration, Ex: 716 nm. (d) Zeta potentials of GNRs, GNR@SiO₂, GNR@SiO₂-NH₂, and GNR@SiO₂-ICG at pH 7.4.

Representative fluorescence emission spectra of GNR@SiO₂-ICG and equivalently free ICG-NHS were shown in Figure 2(c). The fluorescence intensity of GNR@SiO₂-ICG is nearly half of free ICG-NHS. The results suggest that the phenomenon of FRET between GNRs and ICG molecules was still existing despite separating them with a thicker silica layer [22]. Zeta potentials of GNRs, GNR@SiO₂, GNR@SiO₂-NH₂, and GNR@SiO₂-ICG were recorded at pH 7.4 and shown in Figure 2(d). The zeta potential of GNRs was  mV due to the existence of CTAB. Since the existence of –OH groups on the surface of silica shells, the zeta potential of GNR@SiO₂ became  mV. The zeta potential of GNR@SiO₂-NH₂ was  mV, on account of amino groups on the surface of GNR@SiO₂-NH₂. Moreover, after being coupled with ICG-NHS, the zeta potential of GNR@SiO₂-ICG changed into  mV. The potential variations further suggest that the surface of GNR@SiO₂ has been successfully modificated by amine group and coupled with ICG molecules.

3.2. Biocompatibility, Cell Uptake, and Localization of GNR@SiO₂-ICG

To exploit its potential biomedical applications, the biocompatibility assessment in vitro of GNR@SiO₂-ICG was determined by the standard protocol of CCK-8 assay (Figure 4(a)). As expected, GNRs have shown dose-dependent cytotoxicity because of the unbound or loose CTAB molecules [23]. However, after being coated with silica shells, obviously reduced cytotoxicity was observed, which imply that the residual CTAB could not dissociate from nanoparticles [24]. It is worth noting that the viability of A375 cells treated with GNR@SiO₂-NH₂ and GNR@SiO₂-ICG is more than 90% even if the Au concentration reached up to 80 μg/mL. These results clearly indicate that the newly developed nanoprobes possess low cytotoxicity and excellent biocompatibility. Therefore, it is believed that GNR@SiO₂-ICG are useful and promising for applications in biomedicine fields.

The cellular uptake and localization of GNR@SiO₂-ICG were further investigated by bio-TEM, the most direct characterization method. Compared with the untreated control cells (Figure 3(a) and correspondingly higher magnification of Figures 3(b) and 3(c)), Figure 3(d) has shown part of GNR@SiO₂-ICG gathered near the cell membrane and many of them were trapped into the cytosolic vesicles, such as endosomes and lysosomes, indicating that the nanoprobes are internalized into A375 cells via the endocytic pathway. Figures 3(e) and 3(f) have shown higher magnification; the morphological structure of GNRs was clearly visible and distinct from the cellular component due to their high electron density. All results demonstrate that GNR@SiO₂-ICG show negligible cytotoxic effects and can be taken up effectively and internalized well for further cancer therapy.

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Figure 3: TEM images of A375 cells incubated with 10 μg Au content of GNR@SiO₂-ICG for 24 h. ((a), (b), and (c)) Untreated control A375 cells. Scale bars, 5 μm, 500 nm, and 200 nm, respectively. (d) Red arrows indicate endocytosed nanoparticles. Scale bars, 5 μm. ((e) and (f)) Representative ultrastructures of A375 cells containing GNR@SiO₂-ICG. Scale bars, 500 nm and 200 nm, respectively.

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Figure 4: (a) Dark toxicity of A375 cells incubated with various concentrations (0–80 μg Au/mL) of GNRs, GNR@SiO₂, GNR@SiO₂-NH₂, and GNR@SiO₂-ICG, respectively. (b) The untreated cells were used as control group. Scale bars, 20 μm. (c) Two-photon luminescence image of GNRs in A375 cells treated with GNR@SiO₂-ICG (10 μg Au/mL content) for 24 h. Scale bars, 20 μm. Ex: 800 nm. (d) Fluorescence image of ICG in A375 cells incubated with GNR@SiO₂-ICG (10 μg Au/mL content) for 24 h. Scale bars, 20 μm. Ex: 655 nm.

3.3. TPL Imaging and NIR Fluorescence Imaging In Vitro

Two-photon luminescence (TPL) has been particularly appealing for nonlinear optical imaging in biomedical applications due to 3D spatial resolution, low photodamage to target cell/tissue, little near-infrared (NIR) absorption from endogenous species and water, and large penetration depth [12]. However, a serial process involving a sequential photons absorption for creating electrons and holes in the sp-bands and d-bands and then electrons near the Fermi surface recombining with the holes near the X and L symmetry points leads to the observed TPL [25]. In the study, A375 cells were incubated with GNR@SiO₂-ICG for 24 h and imaged by CLSM with excitation wavelength at 800 nm. The untreated cells served as control group. Figures 4(b) and 4(c) presented the overlaid image of bright-field and TPL. It can be seen that the luminescence signal of GNR@SiO₂-ICG incubated cells was relatively uniform, distributed throughout most of cellular cytoplasm. The results substantiate previous research, in which TPL efficiency will receive the largest enhancement when excitation spectrum is superimposed onto the longitudinal surface plasmon band of GNRs, namely, a plasmon-enhanced two-photon absorption cross section [26].

In order to further demonstrate the cellular uptake effect and NIR fluorescence imaging capacity of GNR@SiO₂-ICG, compared with the untreated control A375 cells (Figure 4(b)), the overlaid image of bright-field and TPEF of cells illustrated that cellular uptake of ICG was really obvious through coupling with GNR after incubating with GNR@SiO₂-ICG for 24 h (Figure 4(d)). Because of nonlinear and two long-wavelength photons’ excitation properties, TPEF imaging possesses the advantages of comparatively deep penetration and low photobleaching rates compared with one-photon excitation. In addition, GNR@SiO₂-ICG compound could avoid ICG molecules nonspecific binding to serum proteins thus enhancing intracellular uptake efficiency [7]. Finally, the GNRs core of GNR@SiO₂-ICG would protect exterior ICG molecules from strong light-induced photobleaching because the light absorption and scattering of GNRs are at least 5-6 orders of magnitude stronger than ICG dye molecules and the LSPR band of GNRs was tuned to overlap with the most exciton band of ICG-NHS [10, 27].

3.4. Antitumor Effect of GNR@SiO₂-ICG In Vitro

To validate the potential of GNR@SiO₂-ICG in photothermal therapy, we measured the temperature elevation of GNR@SiO₂-ICG aqueous solutions under 808 nm laser irradiation with an output power density of 1.86 W/cm² for 8 min. Average change in solution temperature for each group was plotted (Figure 5(a)). The heat generation of GNR@SiO₂-ICG increased rapidly in the first three minutes and then rised gradually during the whole irradiation process. The temperature elevation is sufficient to induce cancer cell damage. By contrast, the temperature-rising curve of GNR@SiO₂-NH₂ showed a similar trend but a relatively lower temperature elevation under the same concentration, volume, and NIR irradiation conditions. Nevertheless, the change in temperature of PBS solution was minimal. The results indicate that the composite nanomaterial could rapidly and efficiently convert NIR light into thermal energy. This is attributed to the GNRs core; the strong surface plasmon resonance (SPR) in the NIR region leads to strong photo-thermal conversion effect. Meanwhile, the surrounding tissues will rapidly and effectively convert light energy into thermal energy when the near-infrared chromophore ICG emits photons during the process of returning from an excited state to ground state [15]. Therefore, GNR@SiO₂-ICG possess the synergistic photothermal effect of GNRs and ICG, thus can make better use for cancer therapy.

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Figure 5: (a) Temperature-rising curves of GNR@SiO₂-ICG (red) or PBS (black) in the aqueous solutions exposed to 808 nm laser for 8 min. (b) Phototherapies effect of A375 cells after different treatment and irradiation. (c) Confocal fluorescence images of calcein AM (green, live cells) and PI (red, dead cells) costained cells after phototherapies ablation with the 808 nm laser for 3 min.

To explore the synergistic phototherapies for cancer cells, A375 cells were incubated with GNR@SiO₂-NH₂ or GNR@SiO₂-ICG for 24 h and subdivided into groups with or without NIR laser exposure, respectively. The viability of cells was determined by the CCK-8 assay and the untreated group was assumed to be 100%. As can be seen in Figure 5(b), cells treated with GNR@SiO₂-NH₂ or GNR@SiO₂-ICG and irradiated by NIR laser resulted in a time-dependent inhibition of cell viability. After irradiation for 3 min at a power density of 1.86 W/cm², effective photo-induced cell destruction (>98%) was observed in the cells treated with GNR@SiO₂-ICG, compared with GNR@SiO₂-NH₂ group (91.3%) and untreated control group (12%). The higher cell killing efficiency with GNR@SiO₂-ICG could be attributed mainly to the sufficient amount of heat generation, following passing the heat to the surrounding medium via phonon-phonon relaxation, which leads to irreversible cell destruction through protein coagulation and denaturation as well as cell membrane destruction [28]. In addition, bubble formation around gold nanoparticles is also involved in the case of laser irradiation, which imposes mechanical stress resulting in cell damage [29].

Calcein-AM/PI double staining was employed to further intuitively confirm the effective and specific photo ablation of A375 cells induced by GNR@SiO₂-ICG. All cell images were acquired through confocal fluorescence microscope after being separately incubated with GNR@SiO₂-NH₂ or GNR@SiO₂-ICG for 24 h and irradiated by NIR laser for 3 min, following staining by Calcein-AM and PI. Viable cells cleave Calcein-AM to calcein, thus producing green fluorescence, whereas dead or later apoptosis cells take up PI into the nucleus, resulting in intense red fluorescence [30]. It was found that cells treated with GNR@SiO₂-ICG were effectively killed while GNR@SiO₂-NH₂ treated cells were relatively less destroyed after NIR laser irradiation. As the negative control, no significant cell damage was observed after the same NIR laser irradiation (Figure 5(c)). The results clearly demonstrate that NIR fluorescent dye ICG could remarkably improve the photothermal therapy effect of GNRs and play an important role in synergistic therapeutic effect by photodynamic effect, which is consistent with the cytotoxicity results of phototherapies therapy.

4. Conclusions

In summary, chromophore-enhanced phototherapies killing of tumor cells by laser has generated interest in recent years. In this work, we have successfully prepared a novel ICG-containing nanostructure GNR@SiO₂-ICG that served as an efficient therapeutic agent for cancer cell photo-imaging and phototherapy. Characterization and the study in vitro show that the nanoprobes have the superiority of low cytotoxicity and high chemical stability and biocompatibility. LSPR band of GNRs was overlapped with the most exciton band of ICG-NHS, in which the absorption coefficient of coupled ICG-NHS could be increased. Simultaneously, ICG-NHS will be protected from strong light-induced photobleaching due to the strong optical absorbance cross sections of the GNRs. Our findings suggest that GNR@SiO₂-ICG possess fantastic functionalities of simultaneous TPL imaging mediated by GNRs core and NIR fluorescence imaging induced by ICG dyes. The LSPR peak of GNRs can be tuned to NIR region where light penetration is sufficient depth and intensity for localized photothermal therapy. In addition, ICG molecules were stabilized and delivered by silica-coated gold nanocarrier, thus guaranteeing sufficient ICG and GNRs were internalized into A375 cells via endocytic pathway and passively accumulated at the tumor site via EPR effect. In vitro antitumor efficiency experiments demonstrate that GNR@SiO₂-ICG show superior antitumor effect than GNRs alone. Therefore, GNR@SiO₂-ICG hold significant promise in the development of a new generation of light absorber nanoprobes for phototherapies.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work is supported by the National Natural Scientific Fund (81272987) and Zhejiang Provincial Natural Science Foundation of China (LY12H11011).

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