Synthesis, characterization and application of gold nanoshells using mesoporous silica core

Microporous and Mesoporous Materials 190 (2014) 197–207

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Synthesis, characterization and application of gold nanoshells using mesoporous silica core Nihal Elbialy ⇑, Noha Mohamed, Ahmed Soltan Monem Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

a r t i c l e

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Article history: Received 14 October 2013 Received in revised form 18 December 2013 Accepted 2 February 2014 Available online 12 February 2014 Keywords: Gold nanoshells Mesoporous silica Doxorubicin Photo heat conversion Passive targeting

a b s t r a c t The present study developed a novel multifunctional nanoparticle capable of being targeted passively to the tumor site, mediating sustained drug release as well as providing photothermal therapy. This fabricated nanoparticle is mesoporous silica-loaded doxorubicin covered with a thin layer of pegylated gold (PEG-DOX-MPS-GNSs). The prepared nanoparticles were characterized using transmission electron microscopy, energy dispersive X-ray analysis, UV–VIS absorption spectroscopy, dynamic light scattering, zeta potential measurements and small angle X-ray diffraction. The prepared mesoporous silica nanoparticles (MPS) were approximately 150 nm in diameter and were characterized by its well-ordered mesoporosity of d-spacing 4.5 nm, which enabled a high doxorubicin-loading capacity. Laser scanning confocal microscopy was used to study the dynamics and cellular uptake of PEG-DOX-MPS-GNSs, in addition to its therapeutic efficiency upon NIR irradiation. Superior cytotoxicity in MCF-7 cells was obtained for irradiated PEG-DOX-MPS-GNSs compared with other experimental groups. Intravenous application of PEG-DOX-MPS-GNSs (1 mg/kg), followed by NIR irradiation of the tumor area, inhibited the growth of subcutaneous Ehrlich carcinoma in vivo (p < 0.0001) and induced a stronger anticancer effect compared to other applied oncological modalities. Moreover, histopathological examination demonstrated a high percentage of necrosis in PEG-DOX-MPS-GNSs-treated group (97%) compared with NIR (34%) or control (18%) groups, which was consistent with the in vitro and in vivo findings. Thus, in this context, we present a novel strategy for preparing a photothermal responsive formulation (PEG-DOX-MPS-GNSs), demonstrating the controlled DOX-release behavior and its therapeutic effect. These prepared multifunctional nanoparticles can efficiently convert laser energy into heat, which in turn induces thermal damage and delivery of doxorubicin to the tumor site with a subsequent high therapeutic efficacy. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction In recent years, numerous nanosized drug-carriers have been developed to improve delivery of anticancer drugs to target sites. These drugs, either attached to the surface or encapsulated within carriers, are usually released at target sites by activation of triggering motifs, including pH [1], temperature [2] or enzyme availability [3]. Doxorubicin hydrochloride is a widely used drug for the treatment of different types of cancers, such as leukemia, breast cancer, ovarian cancer, various lymphomas, etc. However, its clinical application is limited by its harmful side effects, the most significant of which is cardiac toxicity, which can result in cardiomyopathy and congestive heart failure [4]. Thus, efforts have been made to develop new drug delivery techniques to reduce these ⇑ Corresponding author. Tel.: +20 235676830 (Office)/222873230 (Home), mobile: +20 1001200674. E-mail address: [email protected] (N. Elbialy). http://dx.doi.org/10.1016/j.micromeso.2014.02.003 1387-1811/Ó 2014 Elsevier Inc. All rights reserved.

undesirable side effects, which could alter the biodistribution of the drug, enhance its deposition at the tumor sites, and improve its therapeutic efficacy [5]. Many drug delivery systems have been studied, such as biodegradable polymers [6], liposomes [7], gold nanoparticles [8], hydrogels [2] mesoporous silica nanoparticles [9,10] and others [11,12]. A successful drug carrier system would be a system that has the smallest drug dosage, could be administrated once and could be released in a controlled manner. Mesoporous silica nanoparticle has been widely investigated, due to its extraordinary chemical and physical properties, e.g. tunable particle and pore size, large specific surface area, high chemical and thermal stability, excellent biocompatibility, and versatile chemistry for further functionalization [13,14]. Further, its pore walls have a high surface density of silanol groups, which could be reactive toward specific guest molecules [15]. Physiological media (water, blood, and tissue) are relatively transparent in the near infrared (NIR) region of the spectrum,

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enabling tissue penetration depths of up to 10 cm [16]. Within the past decade, a wide variety of nanoparticles with high absorption cross sections in the near-infrared have been developed [16–20]. The most readily used in biomedical applications are gold nanocages [11,21], gold nanoshells [18,19], hollow gold nanospheres [22] and gold nanorods [20,21]. These gold nanoparticles strongly absorb in the NIR and efficiently convert photon energy to heat energy, which produces a local increase in temperature at its accumulation sites [18–22]. These particles are characterized by long circulation times in vivo and are chemically inert and non-toxic [21]. Silica–gold (SiO2–Au) consists of a silica dielectric core, which is surrounded by a gold shell tailored to absorb in the near infrared (NIR) region of the spectrum [18,19]. The intracellular generated heat by photo-heat conversion is not only used in cancer photothermal therapy, but is also triggered in the release of chemo therapeutic drugs when gold nanoshells serve as an anticancer-drug carrier. Thus, it is possible to introduce a promising and novel nanostructure that achieves both chemo- and photothermal therapies. The combination of two oncological modalities will firmly establish an efficient therapeutic mode for cancer treatment [23,24,22,25,26]. The development of such a dual functional system, with each individual function acting in a coordinated way with the other, is critical to optimize the therapeutic efficacy and safety of therapeutic regimes. In this study, we developed a promising category of gold nanoshells with mesoporous silica core-loaded doxorubicin (PEG-DOXMPS-GNSs), which are approximately 170 nm in diameter. These nanoshells enable passive targeting to the tumor site and provide combined cancer chemo-photothermal therapy (CCPT). Upon NIR irradiation, these gold nanoshells convert light energy into heat, which markedly affects the release rate of doxorubicin and efficiently kills cancer cells photothermally. Further, the weak acidic extracellular matrix of tumor tissues offers sustained drug release. 2. Experimental 2.1. Materials Gold (III) chloride (HAuCl43H2O, 99.99%), tetraethyl orthosilicate (TEOS, 99%), cetyltrimethylammonium bromide (CTAB) (99%), potassium carbonate (K2CO3, 99%), sodium citrate dehydrate (HOC(COONa)(CH2COONa)22H2O, 99%), thiolated polyethylene glycol (PEG-SH, MW5000), PRMI 1640, fetal bovine serum, trypsin, sodium chloride, Triton X-100, sodium dodecyl sulfate (SDS), Tris buffer, concentrated HCl and doxorubicin hydrochloride were purchased from Sigma–Aldrich. 3-Aminopropyltriethoxysilane (APTES), solution of 0.1 M sodium hydroxide (NaOH), 2-ethoxyethanol (C2H5OCH2CH2OH, 99%), and sodium borohydride (NaBH4, 98%) were purchased from Merck. Ammonia hydroxide (NH4OH, 28%) was purchased from Fluka. The WST-1 proliferation assay kit was purchased from Cayman Chemical. 2.2. Methods 2.2.1. Preparation of gold nanoshells loaded with doxorubicin (PEGDOX-MPS-GNSs) 2.2.1.1. Preparation of mesoporous silica nanoparticles. Mesoporous silica nanoparticles (MPS) were synthesized using CTAB as the porogen and 2-ethoxyethanol as the co-solvent [14]. Typically, 0.5 g of CTAB was dissolved in 70 ml distilled water, and after complete dissolution, 0.5 ml of ammonia hydroxide (28%) and 30 ml of the co-solvent were added. The mixture was vigorously stirred in a closed vessel at room temperature for 30 min. Then, 2.5 ml of TEOS was dropped into the mixture, which was then vigorously stirred for 24 h. A white precipitate was collected using

centrifugation at 4472g for 15 min, washed 10 times with distilled water. Then, the precipitate were further dispersed in ethanol solution (60 ml) containing concentrated HCl (120 ll) and stirred at 30 °C for 3 h to remove the template (CTAB). This surfactant extraction process was repeated twice to ensure complete removal of CTAB. The template removed mesoporous silica nanoparticles were washed with water for three times then dried at 250 °C for 60 min. 2.2.1.2. Amino functionalization of mesoporous silica nanoparticles. Mesoporous silica nanoparticles were then surface functionalized by grafting with 12 mM 3-aminopropyltriethoxysilane (APTES) at a volume ratio 3:7 under constant heat (80 °C) and vigorous stirring for 1 h. The amine-grafted mesoporous silica nanoparticles were then cooled to room temperature, washed and subjected to at least 7 cycles of centrifugation at 2862g in absolute ethanol and deionized water for 30 min. This process was necessary to ensure that all residual reactants were removed. The amine grafted mesoporous silica nanoparticles (AF-MPS) were then resuspended in deionized water at a concentration of 0.3 g/ml. 2.2.1.3. Preparation of seed solution and Doxorubicin loading. The amine grafted mesoporous silica nanoparticles were seeded with Au(OH)3 nanoparticles on their surfaces by the deposition precipitation method DP process [18,27]. The mixture was vigorously stirred and heated at 70 °C for 30 min until the mild white solution turned a light orange color, indicating successful loading of Au(OH)3 nanoparticles on the amine grafted silica and the formation of gold seeds. The seed solution was then centrifuged at 45g for 60 min and washed with distilled water at least 5 times. The final orange pellets were dispersed in phosphate buffer saline (PBS) at pH 7.4 to obtain a final precursor seed solution volume of 40 ml. Four milligrams of doxorubicin hydrochloride (DOX) was dissolved into 1 ml mesoporous silica seed solution at pH 8, and the suspension was shaken in a dark bottle at 37 °C for 24 h. The drug-loaded MPS seeds were then separated using centrifugation at 4472g for 15 min and washed with PBS several times. The free DOX contents in solution were calculated from the calibration curve at an excitation k of 480 nm and emission k of 585 nm using a spectrofluorometer (Shimadzu, RF 5301pc, Japan). The amount of loaded drug was (2.8 mg of DOX/1 ml MPS seeds) approximately 70% of the loading percentage:

Loading efficiency ð%Þ ¼

Initial amount of DOX  Supernatant free amount of DOX Initial amount of drug

2.2.1.4. Preparation of pegylated gold nanoshells loaded with doxorubicin (PEG-DOX-MPS-GNSs). In growing the shell, the pH of HAuCl4 was adjusted to around 10.1 by addition of 60 mg of K2CO3 to 1.5 ml of 25 mM HAuCl4 diluted in 100 ml of water and allowing the solution to stir in the dark overnight at room temperature for the HAuCl4 to hydrolyze and age to give a colorless gold hydroxide solution which called K-gold. The growth of the GNSs was progressively approached by mixing the DOX-MPS seeds with K-gold at a ratio of 1:200 in the presence of sodium borohydride and sodium citrate. Sodium borohydride was used to reduce the complex gold hydroxide anions in the K-gold onto the Au(OH)3 seeds, while sodium citrate was used to slow the reaction and stabilize the gold nanoshells by acting as a capping agent. This reduction resulted in a nearly immediate color change: red, purple, and blue depending on the shell thickness and its degree of completeness. Gold nanoshells surfaces were coated with polyethylene glycol for intravenous injection, by adding 1 ll of 25 mM PEG-SH (MWT 5000) to each 2 ml of gold nanoshells solution and incubating the mixture for 12 h at 4 °C. The suspension was then centrifuged

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at 952g for 30 min to remove residual PEG-SH from the formulation. Prior to the injection, the solution was centrifuged and the billet was suspended in a sterile 0.9% saline solution. The concentration was then adjusted until its optical density was 2 at 805 nm. Finally, DOX concentration was 120 lg/1 ml GNSs. Preparation steps was summarized in Scheme 1. Samples of the pegylated mesoporous gold nanoshells (PEGMPS-GNSs) were also prepared without the loading of DOX. 2.2.2. Sample characterization The size and morphology of the MPS, MPS seeds and MPS-GNSs were determined using transmission electron microscopy (TEM) (FEI Tecnai G20, Super twin, Double tilt, LaB6 Gun) operating at 200 kV. Energy-dispersive X-ray spectroscopy analysis (EDX) of MPS seeds was performed to confirm the binding of gold ions to the mesoporous silica surface. The absorption spectra of MPS, MPS seeds, DOX-MPS seeds, free DOX, MPS-GNSs and DOX-MPSGNSs were measured using a UV–VIS spectrophotometer (Jenway UV-6420; Barloworld scientific, Essex, UK) in the wavelength range from 400 to 900 nm. The dynamic light scattering apparatus (Zeta Potential/Particle Sizer NICOMP TM 380 ZLS, USA) was used to measure the size distribution of the prepared MPS and PEG-DOX-MPS GNSs. Furthermore, the zeta potential of the MPS, AF-MPS, DOX-MPS seeds, DOX-MPS-GNSs, PEG-DOX-MPS-GNSs were measured in deionized water. Small-angle powder X-ray diffraction (XRD) was used to measure the diffraction pattern of MPS using the XPERT-PRO-PAN analytical, Nether land (operating at 45 kV and 30 mA) with a CuKa radiation (k = 1.54056 Å). The sample was scanned from 0.52° to 9.96° (2h), at a scan step time of 3.00 s and a step size (2h) of 0.04°. A Basic Vector, FT/IR-4100 type A (Fourier Transform Infrared (FTIR)) (Germany) was used for obtaining the infrared spectra of MPS, AF-MPS, MPS seeds, DOX, DOX-MPS seeds, DOX-MPS-GNSs, PEG- DOX-MPS-GNSs and PEG-SH in the range of 4000–400 cm1. 2.2.3. Drug release from DOX-MPS seeds and PEG-DOX-MPS-GNSs Sterilized dialysis bags with a dialyzer molecular-weight cut-off of 12,000 Da were used to perform the drug release experiments. These dialysis bags were soaked overnight in the release medium.

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Two phosphate buffered saline (PBS) solutions of pH 7.4 and pH 5.8 were used as the drug release media to simulate normal blood/tissues and tumor environments, respectively. Half milliliter of DOXMPS seeds (2.8 mg/ml) and PEG-DOX-MPS-GNSs (120 lg/ml) were centrifuged and redispersed into release media, and the solution was placed into the dialysis bags. The sealed dialysis bags were placed into brown bottles, and 40 ml of release media was added to each bottle. These bottles were shaken at a speed of 105 rpm at 37 °C under a light-sealed condition. At specific time intervals, three milliliters of the release media were removed to quantify the concentration of the released drug using a spectrofluorometer. It was then returned to the original release media. The concentrations of the released drug were calculated from the calibration curve at an excitation k of 480 nm and emission k of 585 nm using a spectrofluorometer.

Cumulative release ð%Þ ¼

Amount of DOX released Amount of DOX in the nanoparticles  100%

To evaluate the drug release behavior as a function of NIR exposure and incubation time, eight glass tubes each containing 1.5 ml of PEG-DOX-MPS-GNSs (120 lg/ml) at pH 5.8 were exposed to a NIR laser at different time intervals (5, 10, 15, 20, 30, 40, 50, and 60 min). The release experiments were repeated every 24 h for 5 days using the dialysis bag method, as previously described. 2.2.4. Inoculation of the mice with tumor cells The Ehrlich ascites tumor was chosen as a rapidly growing experimental tumor model where various experimental designs for anticancer agents can be applied [18,28,29]. Ehrlich ascites carcinomas cells were obtained from National Cancer Institute ‘‘NCI’’- Cairo University and were intraperitoneally injected into female balb mice. The ascites fluid was collected on the 7th day after injection. The Ehrlich cells were washed twice and then resuspended in 5 ml saline. Female balb mice (20–25 g in body weight and 6–8 weeks old) were obtained from the animal house of NCI and were injected subcutaneously in their right flanks, where the tumors had developed into a single solid form. Tumor growth was monitored post-inoculation until the desired volume

Scheme 1. Summary of PEG-DOX-MPS-GNSs preparation steps.

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was approximately 0.3–0.6 cm3. All animal procedures and care were performed using the guidelines for the Care and Use of Laboratory Animals and was approved by the Animal Ethics Committee at Cairo University [30]. 2.2.5. Thermal transient measurements Thermal transient measurements of the Ehrlich tumor interstitia were obtained using (Wahl TM-410, K-type, USA) a needle thermocouple. The tip of the thermocouple was positioned at the tumor center-of-mass. Twenty-four hours post-intravenous injection (IV) of 200 ll PEG-MPS-GNSs (case I) and PEG-DOX-MPS-GNSs (case II), tumors (n = 3) were exposed extracorporeally to the near infrared (NIR) laser (continuous-wave (CW), compact infrared 807 nm, single mode crystal laser, 300 mW/cm2, 5 mm spot size) for 1 h. Tumor temperatures were recorded during and following the NIR exposure until achieving the initial tissue temperature. Twenty-four hours post-intravenous injection with 200 ll PBS, tumors (n = 3) exposed to the NIR laser and tumor temperatures was recorded (case III). The temperature change was plotted as a function of the NIR irradiation time. 2.2.6. In vitro cytotoxicity of PEG-DOX-MPS-GNSs The breast cancer cell line MCF-7 and human amnion wish cells were cultured in RPMI 1640 containing 10% fetal bovine serum. Cells were maintained at 37 °C in a humidified and 5% CO2 incubator. For all experiments, cells were harvested using 0.25% trypsin in EDTA and resuspended in fresh medium prior to plating. In vitro cytotoxicity was assessed using the WST-1 Cell Viability and Proliferation assay. MCF-7 cells were seeded into 96-well plates at a density of 100 cells per well. After incubation for 24 h at 37 °C in 100 ll of RPMI 1640 medium containing 10% FBS, 50 ll culture medium was discarded then the cells were treated with 50 ll of PEG-DOX-MPSGNSs (DOX concentration 120 lg/ml) group A and B. Group C and D were treated with 50 ll PEG-MPS-GNSs, group F was treated with free DOX (120 lg/ml) and group E was exposed to the NIR laser with no drug administration. Twenty-four hours post incubation groups A, C and E were exposed to the NIR laser for 60 min. Human amnion wish cells were seeded into 96-well plates at a density of 55,000 cell per well. After incubation for 24 h at 37 °C in 200 ll of RPMI 1640 medium containing 10% FBS, 50 ll culture medium was discarded and the cells were treated with 50 ll of various concentrations of samples (free DOX, PEG-DOX-MPS-GNSs) then cells were incubated for 24 h. Four hours post-treatment, 10 ll of the WST-1 solution was added into each well. The cells were incubated for another 4 h, and the absorbance was monitored at 450 nm on an Elisa micro-plate reader (TECAN). Culture medium without nanoparticles was used as the blank control. The cytotoxicity was expressed as the percentage of the cell viability compared with the blank control. 2.2.7. Laser scanning confocal microscopy Laser scanning confocal microscopy (Zeiss, LSM 510, German) was used to study the dynamics and cellular uptake of PEG-MPSGNSs and PEG-DOX-MPS-GNSs in addition to their therapeutic efficiency upon NIR irradiation. MCF 7 cells were incubated with 500 ll PEG-DOX-MPS-GNSs (120 lg DOX/ml) and PEG-MPS-GNSs, in a glass-bottom dish for 24 h, followed by exposure to the NIR laser for 1 h. Four hours post-treatment, the cells were excited using 488-nm excitation light and the fluorescence emission from DOX was imaged at a wavelength between 565 and 630 nm. The fluorescence yields were obtained by normalizing the integrated fluorescence intensities to the cellular area (as indicated using transmission microscopy). Cell cycle progression was monitored by the flow cytometric measurement of DNA content. Analysis of DNA content in cells stained with propidium iodide was performed

using FACScan (Becton Dickinson, USA). The percentage of cells in each phase of the cell cycle was evaluated using the ModFit software (Cell quest). 2.2.8. In vivo NIR laser photo-thermal therapy As tumors reached the desired volume (0.3–0.6 cm3), the treatment began. Sixty mice were initially used and divided into five groups: A, B, C, D and E. Prior to treatment, the mice were anesthetized via an intraperitoneal injection with thiopental (48 mg/kg). Mice of group A were Intravenously injected with 200 ll PEGDOX-MPS-GNSs (1 mg/kg) via the tail vein, and after 24 h the tumor was exposed extracorporeally to the NIR laser for 60 min. Mice in group B were intravenously injected with 200 ll PEG-MPS-GNSs, and the same previous conditions were performed as group A. Mice in group C were intravenously injected with 40 ll of free DOX (4 mg/kg) (therapeutic dose of free doxorubicin) [31]. The skin at the tumor site for groups A, B and D was shaved to maximize the radiation transmittance to the target area. Mice in group D (positive control) were intravenously injected with 200 ll PBS, pH 7.4 and followed the same irradiation conditions as group A and B. Mice in group E (negative control) received no injections (neither GNSs nor buffer) or subsequent laser irradiation. 2.2.9. Tumor size measurements Due to the high growth rate in the Ehrlich tumor model, changes in the tumor volume (DV) were monitored over a 21-day period for the five groups (A, B, C, D and E). The ellipsoidal tumor volume (V) was assessed every 3 days and calculated using the formula V ¼ ðp=6ÞðdÞ2ðDÞ, where D and d represent the long and short axes, respectively, as measured with a digital caliper (accuracy 0.01 mm). The statistical evaluation of the tumor size data was performed using Fisher’s LSD (least significance difference) multiple-comparison test. A p-value less than 0.05 was considered statistically significant. Each data point was represented as the mean ± standard error (SE). In addition, SPSS version 17 was used for the statistical analyses. 2.2.10. Histopathological examination Treatment groups A, B and C were sacrificed immediately after laser exposure to investigate the percentage of tumor cell necrosis. The tumors were excised, fixed in 10% neutral formalin, embedded in paraffin blocks and then sectioned. Tissue sections were obtained directly after treatment and stained with hematoxylin and eosin (H&E). Previous procedures were repeated for the control group E. All tissue sections were examined using a light microscope (CX31 Olympus microscope) that was connected with a digital camera (Canon). 2.2.11. Quantitative determination of the amount of doxorubicin in tumor tissue The accumulated DOX in tumor tissues were assessed for PEG-DOX-MPS-GNSs, and free doxorubicin (1 mg/kg). Tumor bearing mice (n = 3/time point) were intravenously injected with PEG-DOX-MPS-GNSs and free doxorubicin, then tumors were collected at 3, 6, 24, 48 and 72 h. Then, 0.1 g of tumor was homogenized in 1 ml lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.1% SDS and 50 mM Tris, pH 8.0). Tissue lysate were centrifuged at 2000g for 10 min. The supernatant were then isolated and quantitative analysis of DOX was performed using spectrofluorometer at slit width 15. The concentrations of DOX were calculated from the calibration curve at an excitation k of 480 nm and emission k of 585 nm. The final doxorubicin concentrations were expressed as the amount of DOX with nanogram per gram of tissue.

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(a)

Intensity (cts)

500

(b)

400 300

50 nm

200 100 0 0

1

2

3

4

5

2θ (degree) Fig. 1. Small angle X-ray diffraction pattern of the MPS nanoparticles (a) and its TEM image (b).

3. Results and discussion Mesoporous silica nanoparticles (MPS), amine functionalized mesoporous silica nanoparticles (AF-MPS), mesoporous silica seeds (MPS seeds), doxorubicin-loaded mesoporous silica seeds (DOX-MPS seeds), gold nanoshells (MPS-GNSs), pegylated gold nanoshells (PEG-MPS-GNSs) and pegylated doxorubicin-loaded gold nanoshells (PEG-DOX-MPS-GNSs) were characterized using several techniques. Small-angle X-ray diffraction analysis was used to confirm the formation of the ordered mesoporous silica structure. The diffraction pattern of the prepared MPS showed a narrow and strong peak at 2h = 1.94° with d spacing of 45.3 Å indicating a well-ordered mesoporosity (Fig. 1a). TEM images revealed that the MPS nanoparticles exhibit channel-like pores on their surface (Fig. 1b). In addition, the mesoporous silica nanoparticles appeared to be fully covered with small gold particles (Fig. 2a).

To confirm the binding of gold ions to the mesoporous silica surface, Energy-Dispersive X-ray spectroscopy analysis (EDX) of the MPS seeds was performed. The EDX spectrum of the MPS seeds showed the main constituents of MPS, including silicon, oxygen and gold (Fig. 2b). The detected signals of copper and carbon were attributed to the TEM grid while the appearance of the chloride signal was due to the use of gold chloride trihydrate during the preparation of the MPS seeds. The morphology of the final formed gold nanoshell on the mesoporous silica template clearly showed the formation of a uniform gold shell (Fig. 3a and b). FTIR spectroscopic measurements were carried out to investigate the removal of surfactant CTAB (Fig. 4). Owing to the large amount of CTAB exist in the channel, CTAB coated MPS showed the characteristic C–H stretching vibrations at 2925 and 2856 cm1 and C–H deformation vibration around 1475 cm1 (Fig. 4a). After the removal of CTAB, the characteristics CTAB peaks disappeared, suggesting the successful removal of CTAB (Fig. 4b) [32,33]. Using dynamic light scattering, the measured mean diameters of the MPS nanoparticles and DOX-MPS-GNSs were 152 ± 30 nm and 168 ± 39 nm, respectively, which is appropriate for use as drug delivery vehicles in cancer therapy (Fig. 5a). Particles with a diameter of approximately 200 nm are able to penetrate the cell membrane and enter the cytoplasm via endocytosis [34]. In addition, the size of the PEG-DOX-MPS-GNSs enables the selective accumulation of MPS in the extracellular medium of tumor tissue due to an enhanced permeability and retention effect (EPR) of cancerous tissue [35]. The measured zeta potential of the MPS was +33 mV because of the presence of the CTAB covering the MPS surface. The amino functionalized MPS exhibits a surface charge of +21 mV, and the reduction of positivity was attributed to a replacement of the quaternary amine of CTAB with one amine from APTES during the surface functionalization process. In contrast, the MPS seeds

(b)

(a)

10 nm

Fig. 2. TEM image of the MPS seeds (a), and its Energy Dispersive X-ray (EDX) spectrum.

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201

(b)

200 nm Fig. 3. TEM image of the DOX-MPS-GNSs (a) and a high magnification of the gold shell (b).

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b

a

1475 cm-1 -1

2856 cm 2925 cm-1

4000 3500 3000 2500 2000 1500 1000

500

-1

Wavenumber (cm ) Fig. 4. FTIR Spectrum of MPS nanoparticles (a) and AF-MPS nanoparticles (b).

showed a significant reduction in surface charge (3.5 mV) due to the screening effect of gold layer [36]. These results confirmed the formation of a gold layer around the amino functionalized MPS forming MPS seeds. After growing, a gold shell DOX-MPS-GNSs has an average surface charge of 18 mV. PEG coating of the DOX-MPS-GNSs increased the negativity of the gold nanoshell

surface charge, which achieved a high stability and prevented aggregation [37] (Fig. 5b). The absorption spectrum of the prepared MPS nanoparticles showed a decrease in the optical absorption with an increase in wavelength, indicating that the MPS did not exhibit any surface plasmon resonance. After the attachment of small gold ions on the AF-MPS and the formation of MPS seeds, an optical response appeared at 550 nm. Interestingly, the characteristic peak of doxorubicin appeared at 500 nm when DOX-loaded MPS were formed (Fig. 6a). The MPS-GNSs with K-gold to seed ratio 200:1 were found to have a surface plasmon resonance in the wavelength ranging from 610 to 900 nm (Fig. 6b). When DOX-MPS-GNSs were used in the formation of gold nanoshells, two absorption peaks at (500 nm and 610–900 nm) appeared. These two peaks confirmed the formation of gold nanoshells loaded with doxorubicin. In vitro release profile of doxorubicin from MPS seeds and PEGDOX-MPS-GNSs at pH 7.4 and 5.8 PBS solutions was shown in Fig. 7. The results indicated that DOX release from PEG-DOXMPS-GNSs exhibited a relatively slow profile at pH 7.4 suggesting that gold shell was acting as a structurally stable phase at physiological pH in addition to the strong electrostatic attraction between the DOX molecules and MPS pores which prevent DOX diffusion (Fig. 7b). The ability of PEG-DOX-MPS-GNSs to release DOX during the NIR laser exposure was demonstrated when a suspension of PEG-DOX-MPS-GNSs was irradiated with the NIR laser in vitro at room temperature and at pH 5.8 (simulation of tumor environment) (Fig. 7a). Under this condition, 16.5% of doxorubicin was released after 1 h (exposure time). When the laser beam was switched off, a continuous release of DOX was observed up to 120 h at a pH value of 5.8 and temperature of 37 °C (Fig. 7b). A

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20 10 0

MPS

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Fig. 5. Dynamic light scattering size distribution of the MPS nanoparticles (j), DOX-MPS-GNSs (d) (a), and the average value of the zeta potential for the prepared nanoparticles (b).

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MPS nanoparticles MPS seeds Dox-MPS-seeds

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Fig. 6. UV–VIS absorption spectra of the MPS nanoparticles, MPS seeds, DOX-MPS seeds (a), GNSs, DOX-MPS-GNSs, and free DOX (b).

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Time (min)

Fig. 7. The release percentage of doxorubicin from PEG-DOX-MPS-GNSs as a function of the NIR irradiation time (a). The release of doxorubicin from the MPS seeds at pH7.4 (d), pH 5.8 (N) and from PEG-DOX-MPS-GNSs at pH 5.8 (j).

irradiated gold nanoshells which show the loss of the characteristic NIR absorption band of PEG-DOX-MPS-GNSs (Supporting information). As the pH decrease to 5.8 the electrostatic attraction between the DOX molecules and MPS pores decreases which lead to the sustain release of DOX in this pH. Such marked variation in the release profile at different pH substantiates the pH responsiveness of PEG-DOX-MPS-GNSs and indicated that this novel formulation could release doxorubicin specifically at the tumor sites. The release profile of the drug loaded MPS seeds were higher compared to the PEG-DOX-MPS-GNSs, indicating an important role of the formed gold shell in controlling the amount of released drug (Fig. 7b). The ability of the prepared formulations to convert light into heat was assessed in vivo by measuring the tumor temperature as a function of the NIR exposure time (Fig. 8). Thermal transient curves showed the successful photo-heat conversion of NIR-irradiated GNSs, which accumulated in the tumor tissues. The marked increase in tumor tissue temperature, with an average of 13 °C, indicated that efficient photo-heat conversion was induced by both the PEG-MPS-GNSs and PEG-DOX-MPS-GNSs. As the laser beam was switched off, the temperature decreased to normal tissue temperatures. At temperatures greater than 43 °C, protein denaturation and disruption of the cellular membrane are known to occur and ablation of tumor tissues has been shown in numerous cases [39]. In contrast, mice/tumors injected with PBS and treated with NIR laser exposure showed an average increase in tumor temperature up to 4 °C. To evaluate and compare the cytotoxicity of the PEG-DOXMPS-GNSs and free DOX, the WST-1 assay was used. Cells of groups A and C were incubated with PEG-DOX-MPS-GNSs, and PEG-MPS-GNSs, followed by NIR exposure for 1 h. As observed in

Fig. 8. The change in the Ehrlich tumor interstitial temperature during and following NIR laser irradiation for Case I, Case II and Case III.

rapid release of the DOX molecules from the surface or nearsurface regions was clearly observed from the release profile at pH 5.8. This was attributed to the degradation of the gold shell after the exposure to NIR laser, where irradiation of the gold nanoshells can induce melting, evaporation, and fragmentation of nanoshells [38]. This breakdown of gold shells makes the surface of the mesoporous silica exfoliated, which in turn enables the sustain release of the chemotherapeutic loaded drug. This explanation can be emphasized by the UV–VIS absorption spectrum of NIR

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Fig. 9. Cytotoxicity of PEG-DOX-MPS-GNSs (A), PEG-MPS-GNSs (C) against MCF-7 cells incubated for 24 h followed by NIR irradiation for 1 h. Cells incubated for 24 h with PEG-DOX-MPS-GNSs (B), PEG-MPS-GNSs (D) without NIR irradiation. Cells irradiated with NIR laser irradiation (F), and cells incubated with free DOX (120 mg/ml) for 24 h (E) (a). Cytotoxicity of PEG-DOX-MPS-GNSs and free DOX against human amnion wish cells (b).

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(a)

(b)

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Fig. 10. Confocal scanning microscopy images for the MCF7 cell line, control MCF7 cells (a), high magnification of the control cells (b), cells treated with the NIR laser for 1 h (c), cells incubated with PEG-MPS-GNSs for 24 h followed by NIR exposure for 1 h (d) , cells incubated with PEG-DOX-MPS-GNSs for 24 h followed by NIR exposure for 1 h (e) and the fluorescence field of ‘‘e’’ (f).

Fig. 8, PEG-DOX-MPS-GNSs showed the highest cytotoxicity (cell viability percent of 2.53) against MCF7, compared to either PEGMPS-GNSs (cell viability percent of 33.8) or free DOX (group F) (cell viability percent of 80.15). Also, cells incubated with PEG-DOXMPS-GNSs (group B), and PEG-MPS-GNSs (group D) without NIR irradiation show cell viability percent of 68, 97.6, respectively. Moreover, the positive control cells (group E) exposed to the NIR laser for 1 h showed a cell viability of 94%. This rate might be attributed to the therapeutic effect of the released anticancer drug (doxorubicin) out of PEG-DOX-MPS-GNSs during laser exposure which in turn decreased the surviving fraction to 2.53% (Fig. 9a). To assess the cytotoxicity of PEG-DOX-MPS-GNSs on normal cells, human amnion wish cells were exposed to different concentrations of doxorubicin (6–10–12 lM) as free drug and PEG-DOX-MPS-GNSs for 24 h and cell viability were measured. PEG-DOX-MPS-GNSs have no effect on the viability compared to free drug with the same concentration. These results confirm the selectivity of the prepared nanoparticles (Fig. 9b). Confocal laser scanning microscopy images of MCF7 cells showed the rod like shape of the intact cells with the appearance

of few dead cells (Fig. 10a and b). Similarly, MCF7 cells that were exposed to the NIR laser for 60 min showed few dead cells (Fig. 10c). Meanwhile, MCF7 cells incubated with PEG-MPS-GNSs for 24 h showed high cellular uptake leading to severe damage upon NIR laser exposure for 1 h (Fig. 10d). Fig. 10e and f showed MCF7 cells incubated with 120 lg/ml PEG-DOX-MPS-GNSs for 24 h and treated with the NIR laser for 60 min. The observed red fluorescence indicated the accumulation of PEG-DOX-MPS-GNSs in the cytoplasm without an evidence of entering into the nucleus (Fig. 10f). This confirmed that PEG-DOX-MPS-GNSs have been entered into the cell by endocytosis through the plasma membrane of MCF7 cells. As a consequence, these accumulated nanoparticles convert the NIR light into heat, which not only induced thermal damage of tumor cells but also triggered the release of doxorubicin from PEG-DOX-MPS-GNSs. Next, we assessed the effects of the PEG-DOX-MPS-GNSs on cell cycle progression and cell death by the analysis of DNA content using flow cytometry (Fig. 11). Free DOX arrested MCF7 cells in the S phase of the cell cycle. Cell cycle arrest can trigger specific cellular responses, resulting in apoptotic cell death (Fig. 11b).

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Fig. 11. Changes of DNA content in MCF7 cells, (a) control cells, (b) and (c) cell treated with free DOX and PEG-DOX-MPS-GNSs for 24 h, respectively. Then cells were harvested, stained with propidium iodide, and analyzed on FACScan (Becton Dickinson). The percentage of cells in each phase of the cell cycle was evaluated using the ModFit software.

Fig. 12. The average changes in the Ehrlich tumor volume as a function of time for the treatment groups A, B, C, and D as well as the control group E.

Twenty-four hour post treatment with PEG-DOX-MPS-GNS, all cells appear in sub G1 phase of the cell cycle (Fig. 11c). Apoptotic cells are often distinguished on frequency histograms by their fractional DNA content in sub-G1 phase [40]. So, PEG-DOX-MPS-GNSs induce direct cell cycle blocking effect and cell death. These results emphasize that released DOX was the agent responsible for cell cycle arrest and subsequent apoptosis. Therapeutic efficacy of the developed formulation has been assessed by following up the change in tumor volume over a 21-day period in the five groups. Under our experimental conditions, pronounced inhibition in tumor growth was demonstrated in the two animal groups, A and B, compared with the control group E (p < 0.0001 and p < 0.0001, respectively). Group C, administrated with the therapeutic dose of doxorubicin, showed a slight decrease

in tumor volume at day 3, followed by a rapid growth throughout the 19-day period. In addition, group D showed a slight delay in the tumor growth rate compared with the control group (group E) (Fig. 12). These marked decreases in the Ehrlich tumor volume for groups A and B, treated with the suggested protocol, were attributed to selective hyperthermia of tumor tissues injected intravenously with PEG-DOX-MPS-GNSs followed by NIR exposure. Moreover, group A, which was irradiated with NIR laser, triggered the release of doxorubicin markedly from PEG-DOX-MPS-GNSs. We observed that, after laser irradiation was switched off, DOX release extended over at least five days period which is sufficient to induce a high therapeutic efficacy. Histopathological examination of entire tumor sections for the treated experimental groups revealed marked differences in the cellular features accompanied by varying degrees in the necrosis percentage when compared with the control sections. Tumor sections for the Ehrlich tumor cells were excised from the mice of group E (a), group C (b), group B (c) and group A (d) (Fig. 13). The calculated percentages of necrosis for experimental groups A, B, C, and E were 97%, 69%, 34% and 18%, respectively. The negative control (group E), in which the tumors received neither laser treatment nor GNSs injection, showed a normal necrosis percentage of focal and diffuse necrosis (thin & bold arrows, respectively). The former appears as scattered necro-apoptotic bodies within the groups of viable cells, while the latter appears as islands of coagulative necrosis (geographic distribution) showing the ghosts of cells. Hemorrhagic necrosis was also observed (Lower Rt, ‘‘encircled’’), where the mean field count was approximately 18 (Fig. 13a). For the positive control (group C), microscopic examination revealed an increase in the necrosis percentage by up to 34%, with diffuse cellular affection and geographic appearance. The necrotic regions exhibited scattered nuclear karyorrhectic debris and apoptotic bodies (Fig. 13b). This mild cell coagulative necrosis could be attributed to a deep penetrative power of the NIR laser

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(a)

(b)

(c)

(d)

Doxorubicin concentration (ng/g)

Fig. 13. Sections of Ehrlich tumor cells excised from group E (a), e (b), B (c) and A (d) tumor tissues stained with H&E and quantification of the percentage of necrosis. The mean necrosis field counts were 18%, 34%, 69%, 97% for groups E, C, B and A, respectively.

3 hr 6 hr 24 hr 48 hr 72 hr

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Fig. 14. The concentration of DOX in tumor tissue after intravenous administration of free DOX and PEG-DOX-MPS-GNSs.

To quantify and compare the amount of DOX in tumor after intravenous administration of free DOX and PEG-DOX-MPS-GNSs, the concentration of DOX in tumor tissue lysate was measured. Six hours post injection high accumulation of DOX for PEG-DOXMPS-GNSs was maintained, in tumor tissues, over a period of 72 h compared to free DOX. This marked retention of DOX is due to the lake of lymphatic drainage in tumor and the ability of tumor tissue to retain the accumulated particles (EPR effect [35] (Fig. 14). PEG-DOX-MPS-GNSs is an excellent photothermal agent candidate that initiates sustained release. Using this drug carrier, the overall drug consumption and side effects could be significantly reduced. Importantly, this nanocarrier has the ability to be localized at the tumor site to release the loaded drug in a controlled manner. Both the in vitro and in vivo studies suggested that this dual function of gold nanoshells demonstrate an enhanced potential to kill cancer cells compared to both photothermal therapy and chemotherapy alone. 4. Conclusions

beneath the skin. Treatment group B exhibited a tumor field of moderate necrosis with a mean field count of approximately 69%, with viable cells (arrows), pre-necrotic degenerative changes in the vacuolated tumor cells (hydropic change) and indistinct nuclei (encircled). The apoptotic and karyorrhechtic bodies were also increased (Fig. 13c). Treatment group A showed an area of total tumor necrosis (99%). The viable forms show the end stage degeneration of cellular shrinkage and nuclear pycnosis (arrows). The focal groups of neoplastic cells revealed evidence of cytolysis and microcystic change (encircled). Nuclear debris was observed at a lower Rt. zone (Fig. 13d). The upper left island of viable tumor cells showed apoptotic bodies. Histopathological examinations confirmed the observed inhibition of tumor growth rate in the treatment groups compared with the control groups. It can be emphasized from the above results that the doxorubicin released inside the cell lead to apoptotic cell death in addition to heat released induces necrotic cell death.

This study reports a simple method for the preparation of doxorubicin-loaded mesoporous silica. This formulation provides two oncological modalities: photothermal therapy and chemotherapy. The promising DOX-PEG-MPS-GNSs displayed a high potential for therapeutic treatment against MCF7 (in vitro) and Ehrlich carcinoma (in vivo). Furthermore, DOX-PEG-MPS-GNSs also showed an enhanced cellular uptake. Interestingly, this passively targeted nanocarrier was capable of converting the NIR laser into heat, which not only induced tumor cell damage but also triggered drug release with high therapeutic efficacy. Acknowledgments The authors gratefully acknowledge Dr. Tarek El-Bolkini, National Cancer Institute (NCI) – Cairo University for his help in

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the histopathological examination. In addition, we thank Dr. Taher Salah, at the Nanotechnology Characterization Center at the Agriculture Research Center for his help with the confocal laser scanning microscope imaging. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micromeso.2014. 02.003. References [1] D. Schmaljohann, Adv. Drug Deliv. Rev. 58 (2006) 1655–1670. [2] M. Bikram, A.M. Gobin, R.E. Whitmire, J.L. West, J. Control Release 123 (2007) 219–227. [3] R. Goldbart, T. Traitel, S.A. Lapidot, J. Kost, Polym. Adv. Technol. 13 (2002) 1006–1018. [4] Y. Chen, Y. Wan, Y. Wang, H. Zhang, Z. Jiao, Int. J. Nanomed. 6 (2011) 2321– 2326. [5] S. Ibsen, E. Zahavy, W. Wrasdilo, M. Berns, M. Chan, S. Esener, Pharm. Res. 27 (2010) 1848–1860. [6] S. Aggarwal, A. Goel, S. Singla, Adv. Polym. Sci. Technol. Int. J. 2 (2012) 1–15. [7] G.N.C. Chiu, S.A. Abraham, L.M. Ickenstein, R. Ng, G. Karlsson, K. Edwards, E.K. Wasan, M.B. Bally, J. Control Release 104 (2005) 271–288. [8] F. Wang, Y.C. Wang, S. Dou, M.H. Xiong, T.M. Sun, J. Wang, ACS Nano 5 (2011) 3679–3692. [9] A.S. Al-Kady, M. Gaber, M.M. Hussein, E.M. Ebeid, Eur. J. Pharm. Biopharm. 77 (2011) 66–74. [10] Q. He, Y. Gao, L. Zhang, Z. Zhang, F. Gao, X. Ji, Y. Li, J. Shi, Biomaterials 32 (2011) 7711–7720. [11] M.S. Yavuz, Y. Cheng, J. Chen, C.M. Cobley, Q. Zhang, M. Rycenga, J. Xie, C. Kim, K.H. Song, A.G. Schwartz, L.V. Wang, Y. Xia, Nat. Mater. 8 (2009) 935–939. [12] A. Hekmat, A.A. Saboury, A. Divsalar, J. Biomed. Nanotechnol. 8 (2012) 968– 982. [13] Y.S. Lin, K.R. Hurley, C.L. Haynes, J. Phys. Chem. Lett. 3 (2012) 364–374. [14] S. Tan, Q. Wu, J. Wang, Y. Wang, X. Liu, K. Sui, X. Deng, H. Wang, M. Wu, Microporous Mesoporous Mater. 142 (2011) 601–608. [15] A. Sousa, K.C. Souza, E.M.B. Sousa, Acta Biomater. 4 (2008) 671–679. [16] B.G. Prevo, S.A. Esakoff, A. Mikhailovsky, J.A. Zasadzinski, Small 4 (2009) 1183– 1195.

207

[17] P. Rai, S. Mallidi, X. Zheng, R. Rahmanzadeh, Y. Mir, S. Elrington, A. Khurshid, T. Hasan, Adv. Drug Deliv. Rev. 62 (2010) 1094–1124. [18] N. Elbialy, N. Mohamed, A.S. Monem, J. Biomed. Nanotechnol. 9 (2013) 158– 166. [19] J.G. Morton, E.S. Day, N.J. Halas, J.L. West, in: S.R. Grobmyer (Ed.), Methods Mol. Biol., 624, Houston, TX, USA, 2010, pp. 101–117. [20] B. Dickerson, E.C. Dreaden, X. Huang, I.H. El-Sayed, H. Chu, S. Pushpanketh, J.F. McDonald, M.A. El-Sayed, Cancer Lett. 269 (2008) 57–66. [21] S. Jelveh, D.B. Chithrani, Cancer 3 (2011) 1081–1110. [22] J. You, R. Zhang, G. Zhang, M. Zhong, Y. Liu, C.S. Van Pelt, D. Liang, W. Wei, A.K. Sood, C. Li, J. Control Release 158 (2012) 319–328. [23] H. Liu, T. Liu, H. Wang, L. Li, L. Tan, C. Fu, G. Nie, D. Chen, F. Tanga, Biomaterials 34 (2013) 6967–6975. [24] H.J. Lee, Y. Liu, J. Zhao, M. Zhou, R.R. Bouchard, T. Mitcham, M. Wallace, R.J. Stafford, C. Li, S. Gupta, M.P. Melancon, J. Control Release 172 (2013) 152–158. [25] S. Shen, H. Tang, X. Zhang, J. Ren, Z. Pang, D. Wang, H. Gao, Y. Qian, X. Jiang, Biomaterials 34 (2013) 3150–3158. [26] P. Rai, S. Mallidi, X. Zheng, R. Rahmanzadeh, Y. Mir, S. Elrington, A. Khurshid, Adv. Drug Deliv. Rev. 62 (2010) 1094–1124. [27] J.C.Y. Kah, N. Phonthammachai, R. Wan, C. Sheppard, M. Olivo, Gold Bullins 41 (1) (2008). [28] N. Elbialy, M. Abdelhamid, T. Youssef, J. Biomed. Nanotechnol. 6 (2010) 687– 693. [29] O.I. Dasyukevich, G.I. Solyanikn, Exp. Oncol. 29 (2007) 317–319. [30] National Research Council, Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, 1996. [31] S.A. van Acker, E. Boven, K. Kuiper, D.J. van den Berg, J.A. Grimbergen, K. Kramer, A. Bast, W.J. van der Vijgh, Clin. Cancer Res. 3 (1997) 1747–1754. [32] J. Wang, H. Liu, F. Leng, L. Zheng, J. Yang, W. Wangand, C. Huang, Micropor. Mesopor. Mater. 186 (2014) 187–193. [33] Q.L. Yuan, Q.Q. Tang, D. Yang, J.Z. Zhang, F.Y. Zhang, J.H. Hu, J. Phys. Chem. C 115 (2011) 9926–9932. [34] W. Sun, N. Fang, B.G. Trewyn, M. Tsunoda, I.I. Slowing, V.S.Y. Lin, E.S. Yeung, Anal. Bioanal. Chem. 391 (2008) 2119–2125. [35] K.J. Greish, J. Drug Target. 15 (2007) 457–464. [36] J.B. Zhang, N.K. Balla, C. Gao, C.J.R. Sheppard, L.Y.L. Yung, S. Rehman, J.Y. Teo, S.R. Kulkarni, Y.H. Fu, S.J. Yin, Aust. J. Chem. 65 (2012) 290–298. [37] C.G. England, T. Priest, G. Zhang, X. Sun, D.N. Patel, L.R. McNally, V. van Berkel, A.M. Gobin, H.B. Frieboes, Int. J. Nanomed. 8 (2013) 3603–3617. [38] N. Khlebtsov, G. Akchurin, B. Khlebtsov, G. Akchurin, V. Tuchin, V. Zharov, Laser-induced destruction of gold nanoshells: new weapons in the cell-killing arsenal, SPIE Newsroom (2008), http://dx.doi.org/10.1117/2.1200805.1176. [39] D.P. O’Neal, L.R. Hirsch, N.J. Halas, J.D. Payne, J.L. West, Cancer Lett. 209 (2004) 171–176. [40] C.M. Henry, E. Hollville, S.J. Martin, Methods 61 (2013) 90–97.