Surface plasmon-enhanced Ag/CuS nanocomposites for cancer treatment
© Springer-Verlag Wien 2013
Received: 10 April 2013
Accepted: 5 May 2013
Published: 18 May 2013
Photothermal therapy (PTT) for cancer treatment is the use of heat between 41 and 45 °C to damage cancer cells. As a new type of transducer agent for PTT of cancer, CuS nanoparticles have several advantages. The most favorable features are the low cost, simple, and easy preparation and small size for targeting. However, the CuS nanoparticle PTT efficacy needs to be improved for practical applications. In this study, the CuS nano-PTT efficiency is enhanced via the local field enhancement from Ag nanoparticle surface plasmon coupling. The results show that absorbance of CuS nanoparticles in Ag/CuS nanocomposites is enhanced about four times by Ag nanoparticle surface plasmon coupling. Consequently, the PTT efficacy is enhanced and a power of 0.6 W/cm2 with a 980-nm laser is sufficient for Ag/CuS nano-PTT activation for cancer treatment in vitro observations.
Cancer remains one of the most deadly and challenging diseases in the world. In 2008, 7.6 million people died of cancer. Deaths from cancer worldwide are projected to continue rising, with an estimated 13.1 million deaths in 2030, according to the WHO Cancer Fact Sheet (http://www.who.int/mediacentre/factsheets/fs297/en/index.html). To fight the battle of cancer, more and better diagnostic and treatment modalities must be developed.
As a relatively noninvasive and gentle alternative for cancer treatment, hyperthermia is gaining increasing attention (Huang et al. 2006a; Huff et al. 2007). Hyperthermia is commonly defined as heating tissue to a temperature in the range 41–45 °C for tens of minutes (Svaasand et al. 1990; Zee 2002). Tumors are selectively destroyed in this temperature range because of their reduced heat tolerance compared to normal tissue, which is due to their poor blood supply (Huang et al. 2008). On one hand, hyperthermia can induce irreversible cell damage by loosening cell membranes and denaturing proteins (Huff et al. 2007; Huang et al. 2008). On the other hand, as an adjuvant therapy with chemotherapy and radiotherapy, under mild temperature increases, hyperthermia can promote the development of both perfusion and reoxygenation in tumor tissues (Rau et al. 2000; Song et al. 1996), thereby increasing the efficacy of chemotherapy and radiotherapy, respectively. Several methods have been developed for the delivery of thermal energy in a non-topical manner, such as microwave irradiation, radiofrequency pulses, and ultrasound. They can penetrate deep into tissue but may require a high fluence because of their diffuse nature, possibly producing undesirable hyperthermic effects in surrounding tissues (Li and Gu 2010). Laser irradiation at near-infrared (NIR) frequencies can penetrate tissues with sufficient intensity and higher spatial precision for inducing localized hyperthermia (Castrén-Persons et al. 1991; Chen et al. 1997), but effective mechanisms are needed to transduce light energy into heat and to discriminate unhealthy from healthy cells (Huff et al. 2007). One promising approach is to introduce the photothermal agents in the near-infrared region.
Photothermal therapies (PTT) for cancer have been widely investigated as a minimally invasive oncological treatment strategy in which photon energy is converted into heat to induce cellular destruction (O’Neal et al. 2004a; Rozanova and Zhang 2009; Huang and El-Sayed 2011). With the tremendous development of nanotechnology, gold nanostructures such as nanospheres (El-Sayed et al. 2006; Khlebtsov et al. 2006), nanorods (Dickerson et al. 2008; Huang et al. 2006b), nanoshells (O’Neal et al. 2004b; Loo et al. 2005), nanocages (Chen et al. 2005; Hu et al. 2006), as well as carbon nanotubes (Zhou et al. 2009; Robinson et al. 2010) have been investigated extensively for PTT therapy in the near-infrared region. CuS nanoparticles have a broad absorption from 700 to 1,100 nm, and we first demonstrated that the interaction of CuS nanoparticles with near-infrared light can generate heat that can be harnessed for PTT of cancer cells (Li et al. 2010; Zhou et al. 2010). As a new type of agent for photothermal treatment of cancer, CuS nanoparticles have several advantages such as the low cost, simple, and easy preparation and small size for targeting (Li et al. 2010). However, the PTT efficiency using CuS nanoparticles as transducers is low and needs to be improved for practical applications (Tian et al. 2011; Lakshmanan et al. 2012). Here, we investigate the enhancement of CuS nanoparticle PTT efficacy by Ag nanoparticle surface plasmon coupling in Ag/CuS nanocomposites for cancer treatment. A surface plasmon is a localized electromagnetic field at the metal dielectric interface (Englebienne et al. 2003). Surface plasmons can lead to significant electric field enhancement near the surface and thus increase the rate of any processes that depend on the field intensity. The field enhancement due to surface plasmon resonance is predicted to be ∼1,000 times larger than the incident field and, therefore, it can be used to increase any light excitation or transition processes at the interfaces (Aslan et al. 2005; Lakowicz 2001). Because of the localized field amplification that occurs, excitation of surface plasmons in metal nanoparticles placed on a semiconductor might be expected to enhance optical absorption of incident photons within the semiconductor region near each nanoparticle. We have studied and observed the absorption enhancement of CuS by surface plasmon in Au/CuS nanocomposites and the application of Au/CuS for cancer treatment with improved efficacy (Lakshmanan et al. 2012). Here, we report the observations on Ag/CuS nanocomposites for surface plamosn enhancement and the application for Ag/CuS nanocomposites for cancer treatment by in vitro studies. We are interested in Ag/CuS nanocomposites because it has been reported that among the three metals (Ag, Au, Cu) with efficient plasmon resonances, Ag exhibits the highest efficiency of plasmon excitation (Evanoff and Chumanov 2005). Moreover, Ag is much cheaper than Au. Therefore, as photothermal transducer agents for cancer treatment, Ag/CuS nanocomposites might have a higher potential and better market than Au nanostructures and Au/CuS nanocomposites.
2 Materials and methods
Copper(II) chloride dehydrate (CuCl2∙2H2O), sodium sulfide (Na2S), sodium citrate, sodium carbonate (Na2CO3), thiobenzoic acid, copper(II) nitrate hemi(penta hydrate) (Cu(NO3)2∙2.5H2O), silver perchlorate anhydrous (AgClO4), hexadecyltrimethylammonium bromide (CTAB), and l-ascorbic acid were purchased from Sigma-Aldrich (MO, USA). Sodium borohydride (NaBH4) was obtained from Acros Organics (NJ, USA). Nickel nitrate (Ni(NO3)2∙6H2O) was purchased from Alfa Aesar.
2.2 Preparation of Ag nanoparticles
Ag nanoparticles were grown using the seeded growth method according to the literature (Sun et al. 2009). Specifically, the seeds were made by the addition of a freshly prepared, ice-cold 0.01 M NaBH4 solution (600 μL, 0.006 mmol) into a mixture solution composed of 0.01 M AgClO4 (250 μL, 0.0025 mmol) and 0.1 M CTAB (7.5 mL, 0.75 mmol). The resultant solution was mixed by rapid inversion for 2 min and was then kept at room temperature for 1 h before use. The growth solution was prepared by the sequential addition of 0.1 M CTAB (6.4 mL, 0.64 mmol), 0.01 M AgClO4 (800 μL, 0.008 mmol), and 0.1 M ascorbic acid (3.8 mL, 0.38 mmol) into water (32 mL). The CTAB-stabilized seed solution was diluted ten times with water. The diluted seed solution was then added into the growth solution for the growth of Ag nanoparticles. The resultant solution was mixed by gentle inversion for 10 s and then left undisturbed overnight.
2.3 Synthesis of Ag/CuS nanocomposites
Nickel thiobenzoate ((PhCOS)2Ni) is used as the sulfur source for Ag/CuS synthesis and is synthesized based on the synthetic route reported in the literature (Sun et al. 2009). Sodium thiobenzoate was first prepared by mixing together Na2CO3 (211.98 mg, 2.00 mmol), thiobenzoic acid (PhCOSH, 520 μL, 4.40 mmol), and water (1 mL) at room temperature. The resultant concentrated PhCOSNa solution was then added into an aqueous 0.4 M Ni(NO3)2 (10 mL, 2.00 mmol) solution to produce nickel thiobenzoate ((PhCOS)2Ni). The dark red precipitate was washed with water and dried under ambient conditions.
To prepare Ag/CuS nanocomposites, (PhCOS)2Ni (2 mg, 0.006 mmol) was dissolved in the Ag nanoparticle dispersion (10 mL), followed by the addition of 0.01 M Cu(NO3)2 solution. The concentration of Cu2+ in the solution was controlled to be 300 μM. The resultant mixture solution was transferred into an autoclave and heated at 140 °C for 3 h, and then cooled down to room temperature. The prepared nanoparticle solution was dialyzed for 3 h to remove all the unreacted chemicals.
2.4 Preparation of CuS nanoparticles
CuS nanoparticles were prepared as follows: to 100 mL of an aqueous solution containing CuCl2(0.1 mmol, 17.05 mg) and sodium citrate (0.068 mmol, 20 mg), 1 mL of sodium sulfide solution (Na2S, 0.1 M) was added while stirring at room temperature. The pale blue CuCl2 solution turned dark brown immediately upon the addition of sodium sulfide. After 5 min, the reaction mixture was heated to 90 °C and stirred for 15 min, until dark green solution was obtained. The citrate-coated CuS nanoparticles were obtained and stored at 4 °C.
2.5 Characterization of Ag/CuS nanocomposites
Ag/CuS nanocomposites in solution were placed onto holey carbon-covered copper grids for high-resolution transmission electron microscope (HRTEM) observation. The HRTEM images of the particles were obtained with a JEOL JEM-2100 electron microscope (Tokyo, Japan) with accelerating voltage of 200 kV. The absorption spectra were recorded using a Shimadzu UV-2450 UV–Vis spectrophotometer (Kyoto, Japan). The luminescence excitation and emission spectra were measured using a Shimadzu RF-5301PC fluorescence spectrophotometer (Kyoto, Japan). The Ag and Cu element analysis were operated on the PerkinElmer ELAN DRC II ICP Mass Spectrometer system (ICP-MS) with an autosampler.
2.6 Photothermal effect in aqueous solution
For measuring temperature change mediated by CuS nanoparticles or Ag/CuS nanocomposites, 980 nm near-infrared light (Opto Engine LLC) at an output power of 0.6 W/cm2 was delivered through a cuvette containing the nanoparticles (1 mL). The Cu concentration in both Ag/CuS nanocomposites and CuS nanoparticles was 13.912 mg/L. The temperature was measured by a digital thermometer (Fisher Scientific “Traceable”) over a period of 20 min. Water was used as a control.
2.7 In vitro fluorescence imaging
In this study, prostate cancer PC-3 cells were used to evaluate the fluorescence imaging of Ag/CuS nanocomposites. PC-3 were cultured in F-12K medium supplemented with 10 % fetal bovine serum and 1 % penicillin–streptomycin solution, and incubated at 37 °C in a water-saturated incubator with 5 % CO2. The cells were treated with 10 % of original Ag/CuS nanocomposites for 24 h. The Ag and Cu concentration of original Ag/CuS nanoparticles were 46.606 and 13.912 mg/L, respectively. Fluorescence imaging was done on the following day using an Olympus 1X71 inverted fluorescence microscope.
2.8 Time course of cellular uptake by PC-3 cells
The time course of cellular uptake was also carried out on prostate cancer PC-3 cells. Aliquots of the cultured cells were incubated with 5 % of original Ag/CuS nanocomposites for 10 min, 30 min, 1 h, 2 h, 4 h, and 8 h at 37 °C under 5 % CO2 in the 1.5 mL centrifuge tubes. After incubation, the cells were washed two times with PBS. All the imaging results were examined by an Olympus 1X71 Inverted Microscope (Tokyo, Japan).
2.9 In vitro photothermal ablation of cancer cells with Ag/CuS nanocomposites
The cells were cultured as described in the above section and incubated with 2.5, 5, and 10 % of the original Ag/CuS nanoparticle solution at 37 °C and 5 % CO2 for 4 h. After incubation was completed, the culture medium with nanoparticles were removed and resupplied with fresh medium. The cells were then irradiated with a 980-nm NIR laser (Opto Engine LLC) at an output power of 0, 0.6, 1.2, or 1.8 W/cm2 for 5 min. Fluorescence imaging was used to see the number of living and dead cells after photothermal treatment. The live cells were stained with calcein AM and the dead cells with ethidium homodimer-1 (EthD-1), according to the manufacturer’s suggested protocol (Invitrogen). The cells were imaged under an Olympus 1X71 inverted fluorescence microscope.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was also applied to estimate the photothermal effect of Ag/CuS nanocomposites. The cultured PC-3 cells were incubated with 5 and 10 % of original Ag/CuS nanocomposites for 4 h and were irradiated with a 980-nm NIR laser (Opto Engine LLC) at an output power of 0, 0.3, 0.6, or 0.9 W/cm2 for 1 min. Ten microliters of 5 mg/ml MTT solution was added to each well and incubated for 4 h at 37 °C under 5 % CO2. The supernatant was then removed, and the cells were lysed with 50 μL dimethyl sulfoxide. Using a microplate reader, absorbance was recorded at 570 nm to determine cell survival.
3 Results and discussion
Surface plasmon-enhanced photothermal therapy for cancer treatment was investigated in vitro on PC-3 prostate cancer cells using Ag/CuS nanocomposites. The diameters of the Ag/CuS nanocomposites are in the range of 10–20 nm and they have a high absorbance in the near-infrared region. Ag/CuS nanocomposites can get into PC-3 cancer cells and mainly locate in the cytoplasm. The treatment observations found that 5 % of original Ag/CuS nanocomposites activated by a 980 nm laser at 0.6 W/cm2 can efficiently kill cancer cells. With the increase of the nanocomposite concentration or the laser output, more and more cells were killed. Comparing with CuS nanoparticles, surface plasmon-enhanced Ag/CuS nanocomposites have a higher efficiency for the photothermal therapy. It is also a new and promising modality for cancer treatment.
We would like to acknowledge the support from the startup funds from UTA, the NSF and DHS joint ARI program (2011-DN-077-ARI053-03), and the US Army Medical Research Acquisition Activity under contracts of W81XWH-10-1-0279 and W81XWH-10-1-0234. This work was also supported by National Natural Science Foundation of China grant (no. 81072596) and program for New Century Excellent Talents in University (no. NCET-08-0226). At last, we are also grateful for the financial support of China Scholarship Council (no. 2011616067).
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