Coating Urchinlike Gold Nanoparticles with Polypyrrole Thin Shells To

Article pubs.acs.org/Langmuir

Coating Urchinlike Gold Nanoparticles with Polypyrrole Thin Shells To Produce Photothermal Agents with High Stability and Photothermal Transduction Efficiency Jing Li,† Jishu Han,† Tianshu Xu,‡ Changrun Guo,‡ Xinyuan Bu,† Hao Zhang,*,† Liping Wang,*,‡ Hongchen Sun,*,§ and Bai Yang† †

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, ‡College of Life Science, and §School of Stomatology, Jilin University, Changchun, PR China S Supporting Information *

ABSTRACT: Photothermal therapy using inorganic nanoparticles (NPs) is a promising technique for the selective treatment of tumor cells because of their capability to convert the absorbed radiation into heat energy. Although anisotropic gold (Au) NPs present an excellent photothermal effect, the poor structural stability during storage and/or upon laser irradiation still limits their practical application as efficient photothermal agents. With the aim of improving the stability, in this work we adopted biocompatible polypyrrole (PPy) as the shell material for coating urchinlike Au NPs. The experimental results indicate that a several nanometer PPy shell is enough to maintain the structural stability of NPs. In comparison to the bare NPs, PPy-coated NPs exhibit improved structural stability toward storage, heat, pH, and laser irradiation. In addition, the thin shell of PPy also enhances the photothermal transduction efficiency (η) of PPy-coated Au NPs, resulting from the absorption of PPy in the red and near-infrared (NIR) regions. For example, the PPy-coated Au NPs with an Au core diameter of 120 nm and a PPy shell of 6.0 nm exhibit an η of 24.0% at 808 nm, which is much higher than that of bare Au NPs (η = 11.0%). As a primary attempt at photothermal therapy, the PPy-coated Au NPs with a 6.0 nm PPy shell exhibit an 80% death rate of Hela cells under 808 nm NIR laser irradiation.

■

INTRODUCTION Photothermal therapy using inorganic nanoparticles (NPs) with near-infrared (NIR) absorption, 650−980 nm, is a promising technique for the selective treatment of tumor cells.1−6 The NPs are capable of converting light energy into heat, thus making the temperature in the treated volume increase above the thermal damage threshold, deconstructing the tumor cells.7,8 In addition, the specificity of photothermally conducting NPs permits directed cell death without damaging healthy tissues.9−11 The use of NIR light is essential for photothermal therapy because of the minimal absorbance by skin and tissue, thus allowing for noninvasive and deep tissue penetration.12 Among various tested nanomaterials, gold (Au) NPs with diversified morphologies are the most popular photothermal agents because they possess strong optical adsorption in the red and NIR regions, which is tunable via morphology optimization and controlled self-assembly.13−17 Au nanorods, nanoshells, and nanocages are good photothermal agents, as represented by the capability to generate considerable heat energy under NIR irradiation.18−24 As novel Au nanostructures, branched Au NPs also strongly absorb NIR irradiation; meanwhile, the highly branched structures and large specific surface area lead to an even higher photothermal transduction efficiency (η) in © XXXX American Chemical Society

comparison to smooth nanostructures because of the easier penetration of the electric field.25−28 Therefore, branched Au NPs are considered to be competitive candidates within the group of photothermal agents. Structural stability and η are the main issues in employing branched Au NPs in photothermal therapy, which are also problems for other nonspherical Au nanostructures.29−34 The poor structural stability essentially arises from the synthesis mechanism of branched Au NPs in which the growth of branches has to be promoted by kinetics-favored factors.35−38 The as-synthesized branched NPs are thermodynamically unstable, having a great tendency to transform into stable spherical particles or huge aggregates during storage.37−39 Furthermore, upon laser irradiation, the heat generated from the photothermal effect even melts the anisotropic Au nanostructure into spherical particles, thus lowering the photothermal effect.29 The envelopment of NPs by silica and/or polymer shells is efficient at enhancing the stability and subsequently the practicality of nanomaterials.40−44 For Received: April 14, 2013 Revised: May 20, 2013

A

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

amount of acidic (NH4)2S2O8 from 1 to 3 to 5 to 10 mL, whereas the amounts of SDS and pyrrole were both fixed at 600 μL. In all experiments, the volume of urchinlike Au NP aqueous solution was 10.7 mL and the reaction mixture was incubated at room temperature for 7 h. Cell Culture. The human cervical carcinoma cell line (Hela cells) was maintained at a density of 1 × 105 cells/mL in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 humidified atmosphere at 37 °C. In the assay, 100 μL of a cell supernatant is added to each well. Cytotoxicity Assay of PPy-Coated Urchinlike Au NPs. This assay was carried out in 96-well culture plates. Au@PPy NPs were foremost diluted to specific concentrations using RPMI-1640 medium with 10% FBS. Hela cells were seeded on 96-well plates overnight. After the cellular supernatant was discarded, the cells were incubated in 100 μL of different concentrations of Au@PPy solution at 37 °C for 24 h. The plates were analyzed for cell viability using an MTT assay.50 The Au NPs in each concentration were assayed in three wells, and the assay was repeated three times. Photothermal Effect of PPy-Coated Au NPs. The Hela cells were seeded on 96-well plates overnight. After the cellular supernatant was discarded, the cells were incubated in 100 μL of a culture medium with an Au@PPy concentration of 0.125 mg/mL for 4 h. The samples were irradiated with an 808 nm NIR laser with different laser power densities for 3 min. The plates were analyzed for cell viability using an MTT assay. Each laser power density was assayed in three wells and repeated three times. Apoptosis Staining by Hoechst 33342 and Propidium Iodide. The Hela cells were seeded on 12-well plates overnight. After the cellular supernatant was discarded, the cells were incubated in 500 μL of a culture medium with an Au@PPy concentration of 0.125 mg/mL for 4 h. The samples were irradiated with an 808 nm NIR laser with a power density of 1.24 W/cm2 for 10 min. The Hela cells were cultured for 1 h and stained with 0.01 mg/mL Hoechst 33342 and 0.05 mg/mL propidium iodide (PI). Characterization. UV−visible absorption spectra were obtained using a Shimadzu 3600 UV−vis−NIR spectrophotometer at room temperature under ambient conditions. Transmission electron microscopy (TEM) was conducted using a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV with a CCD camera. An (NH4)6MO7O24 aqueous solution (2 w/v%) was also used as the negative stain to improve the contrast. A JEOL JSM-6700F scanning electron microscope (SEM) with a primary electron energy of 3 kV was employed to examine the morphologies of urchinlike Au NPs. Fourier-transform infrared (FTIR) spectra were obtained with a Bruker Vertex 80 V FTIR spectrophotometer. Dynamic light scattering (DLS) measurements were performed using a Zetasizer NanoZS (Malvern Instruments). Bright-field and fluorescent images of Hela cells were observed with an Olympus IX51 inverted fluorescence microscope. In studying the photothermal effect, an 808 nm diode laser (LEO photonics Co. Ltd.) was employed with a laser spot of 1 cm2.

example, Au nanorods can be enveloped with a silica shell, which simultaneously improves the stability of nanorods and also provides the medium for carrying medicines.42 However, this strategy must be restrainedly implemented for the NPs in photothermal therapy because a protective shell may hamper the penetration of the electric field and heat transfer. A proper selection of shell material and the thickness control should be the keys. Ideal shell materials should be inert and biocompatible, simultaneously possessing a photothermal effect that can enhance or at least not lower η. In addition, Au nanomaterials also strongly scatter the irradiation, thus lowering η. Such a loss is expected to be compensated for by choosing shell materials with strong absorption in the red and NIR regions. In this scenario, the polymers with red and NIR absorption are potential candidates for coating Au NPs. Because of the strong absorption in the NIR region, polypyrrole (PPy) possesses a high photothermal transduction efficiency under NIR irradiation.45 Meanwhile, in vivo studies have confirmed that PPy has good biocompatibility.46 These make PPy a promising photothermal agent.47−49 In this work, PPy is selected to coat urchinlike Au NPs to produce composite NPs. The capability to control the thickness of the PPy shell via oxidative polymerization allows the revealing of the thickness effect on the structural stability and the photothermal transduction of the composite NPs. Systematic studies indicate that PPy-coated Au NPs (Au@PPy) exhibit improved structural stability toward storage, heat, pH, and laser irradiation in comparison to bare Au NPs. Resulting from the dual absorbance of Au NPs and the PPy shell, the composite NPs also have a much higher η. As an example, composite NPs with an Au core diameter of 120 nm and a PPy shell of 6.0 nm indicate an η of 24.0% at 808 nm, which is 2 times higher than the η of bare Au NPs. As an attempt at photothermal therapy, PPy-coated Au NPs with a 6.0 nm PPy shell exhibit an 80% death rate of Hela cells under 808 nm NIR laser irradiation.

■

EXPERIMENTAL SECTION

Materials. Pyrrole (99%, Acros) stored at 4 °C, hydrogen tetrachloroaurate(III) (HAuCl4, 99.9%, Alfa Aesar), hydroquinone (98%), sodium citrate (99.0%), dodecyl sodium sulfate (SDS, 86%), (NH4)2S2O8 (98%), (NH4)6MO7O24 (99.0%), HCl, NaOH, and RPMI-1640 medium were analytical grade and used as received. In all experiments, deionized water was used. Preparation of PPy-Coated Urchinlike Au NPs. The urchinlike Au NPs were foremost synthesized through a seed-mediated method following our previous publication (Figures S1 and S2).37 As soon as the urchinlike Au NPs were synthesized, a 10.7 mL aqueous solution of branched NPs, 600 μL of 40 mM SDS, and 600 μL of 10 mM pyrrole aqueous solution were mixed under vigorous stirring. Subsequently, 3 mL of 2 mM acidic (NH4)2S2O8 solution was added. After being stirred for 10 s, the reaction mixture was incubated at room temperature for 7 h to ensure complete polymerization. The PPy-coated NPs were purified by centrifugation at 6500 rpm for 10 min. After the removal of the supernatant, the products were redispersed in deionized water to achieve the suspension with a specific concentration. As observed by TEM, the thickness of the PPy shell was 6.0 nm. The PPy-coated NPs could be stored in water for more than 1 year without morphology variation. Control Experiments. The effect of SDS was studied by altering the amount of SDS aqueous solution from 200 to 600 to 1800 to 2400 μL, whereas the amounts of pyrrole and acidic (NH4)2S2O8 were fixed at 600 μL and 3 mL, respectively. To investigate the effect of pyrrole, we formed the PPy shell by altering the amount of pyrrole aqueous solution from 200 to 600 to 1000 to 1500 μL, whereas the amounts of SDS and acidic (NH4)2S2O8 were fixed at 600 μL and 3 mL, respectively. The effect of (NH4)2S2O8 was studied by altering the

■

RESULTS AND DISCUSSION We have reported the synthesis of urchinlike Au NPs in the presence of hydroquinone through a seed-mediated growth method.37 On the basis of size and morphology control, the absorption spectra of the urchinlike NPs cover the red and NIR regions (Figure S1). Although this property strongly permits application in photothermal therapy, the poor structural stability in the biological tests limits the practical application. To improve the structural stability, we coated the PPy shell on the urchinlike NPs through oxidative polymerization using (NH4)2S2O8 as the initiator. Experimentally, acidic (NH4)2S2O8 was added to the aqueous mixture of urchinlike Au NPs, dodecyl sodium sulfate (SDS), and pyrrole to initiate polymerization. For the preparation of composite NPs with a 6 nm PPy shell, 10.7 mL of an aqueous solution of urchinlike B

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Figure 1. TEM images of the urchinlike Au NPs (a) before and (b) after the PPy coating. (c) DLS size distribution of PPy-coated urchinlike Au NPs. (d) UV−vis spectra of the urchinlike Au NPs before and after the PPy coating.

Au NPs, 600 μL of 40 mM SDS, 600 μL of a 10 mM pyrrole aqueous solution, and 3 mL of a 2 mM acidic (NH4)2S2O8 solution were mixed and stirred at room temperature for 7 h to ensure complete polymerization. The color of the solution gradually turned black, indicating the polymerization of pyrrole. Figure 1b indicates the transmission electron microscopy (TEM) image of the composite NPs. Under TEM, urchinlike Au NPs with diameters of about 120 nm are clearly observed; these are covered with a shell with low contrast. By comparison with the TEM image of the original NPs without a PPy coating (Figure 1a), the shell is assigned to PPy. Because the contrast of polymer materials is usually worse than that of inorganic materials, the (NH4)6MO7O24-stained samples are further characterized by TEM, which clearly presents the PPy shell (Figure S3). The true diameter of PPy-coated NPs in aqueous solution is measured by dynamic light scattering (DLS), which is about 145 nm (Figure 1c). Obviously, the DLS datum point is slightly larger than the TEM result because DLS shows the hydrodynamic diameter of the composite NPs in the aqueous solution rather than that of the dried NPs under TEM. The FTIR spectrum further proves the coating of PPy as well as the presence of a little SDS on the composite NPs (Figure S4). A clear red shift of the UV−vis absorption spectrum of the composite NPs is observed (Figure 1d). This is mainly attributed to the sum of Au and PPy spectra. The formation of an Au−PPy core−shell structure may also contribute to the spectral red shift because the PPy shell alters the localized electric field distribution of urchinlike Au.42 The anisotropic distribution of the electric field on the branches is more favorable. Moreover, the PPy coating is applicable to urchinlike Au NPs with different sizes (Figure S5). In particular, the absorption red shift is more obvious for the composite NPs with a larger Au core, thus greatly favoring the NIR absorption

of Au (Figures S1h and S5g). Note that the composite NPs possess an absorption peak at around 700 nm (Figure 1d). Although the 676 nm laser most closely matches the NPs, 808 nm light is closer to the NIR region. Such NIR light leads to weak autofluorescence and strongly penetrates live tissues, which is welcomed by the current biomedical diagnosis and therapy.51,52 In addition, the composite NPs also have strong absorption at 808 nm (Figure 1d). These inspire us use an 808 nm laser to study the photothermal effect. The thickness of the PPy shell is controllable by altering the concentrations and feed ratio of the materials. Figure 2 displays the effect of SDS concentration on the PPy shell thickness, represented by increasing the amount of 40 mM SDS from 200 to 2400 μL. With the increase in SDS, the shell thickness decreases from 15.6 to 3.5 nm (Figure 2). This trend is attributed to the competitive adsorption of pyrrole on the NPs and in solution, which is determined by the SDS concentration. In the absence of SDS, hydrophobic pyrrole is immiscible with the aqueous solution of urchinlike Au NPs, thus leading to phase separation. When the amount of SDS is increased from 200 to 2000 μL, the zeta potential of the as-prepared composite NPs increases from −41.3 to −53.6 mV (Table S1). This means that the composite NPs are negatively charged by adsorbing SDS; meanwhile, more SDS micelles form in the solution with a greater amount of SDS. SDS is an anionic surfactant, which may simultaneously adsorb on the surface of urchinlike Au NPs and form free micelles in the solution. Because of the large surface area of NPs, the adsorption of SDS on NPs is favorable, whereas free SDS micelles form only with excess SDS. Pyrrole is a cationic monomer. The electrostatic attraction between pyrrole and SDS makes SDS the template to direct the polymerization of pyrrole, either on the NP surface or onSDS micelles.53−55 The competitive polymerization is C

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

As mentioned in our previous report, the as-synthesized urchinlike Au NPs without a PPy coating can maintain the branched structures for 10 days at room temperature. Further storage will lead to the morphology variation toward spherical particles and finally aggregates.37 The morphology evolution might occur through two pathways: the diffusion of Au ions and/or small Au clusters that decompose from the urchinlike NPs.30 Because Au is in the form of Au0, the diffusion of free Au ions should not be the main pathway. Consequently, when the mobility of Au clusters is suppressed, the formation of the PPy shell should greatly enhance the structural stability of urchinlike Au NPs. In our observation, the PPy-coated urchinlike Au NPs can be stored in the solution for more than 1 year without any morphology variation (Figure 3). TEM images indicate that the NPs maintain their original size and branched structures (Figure 3a). UV−vis spectra also exhibit the unchanged absorption property (Figure 3b). The PPycoated urchinlike Au NPs also exhibit improved pH stability (Figures 4 and S8). The original Au NPs are stable only in the

Figure 2. TEM images of PPy-coated urchinlike Au NPs that are prepared through the addition of (a) 200, (b) 600, (c) 1800, and (d) 2400 μL of 40 mM SDS. The amounts of pyrrole and (NH4)2S2O8 are fixed at 600 μL and 3 mL, respectively. Scale bar in the insets: 25 nm.

greatly determined by the SDS concentration. Because most SDS adsorbs on the NPs at a low SDS concentration, it significantly conducts the polymerization of pyrrole on NPs. As a result, a thick PPy shell forms. On the contrary, the increased free SDS micelles in the presence of excess SDS facilitate pyrrole polymerization in the solution, therewith resulting in the thin PPy shell on the NPs. Note that although pyrrole is soluble in water, PPy is very hydrophobic. As a result, the outmost surface layer of the composite NPs is SDS, which contributes to the aqueous dispersibility. The amounts of pyrrole and (NH4)2S2O8 also influence the thickness of the PPy shell, which is not an obvious effect of SDS. As the amount of pyrrole is increased from 200 to 1500 μL, the thickness of the shell gradually increases from 3.5 to 7.8 nm (Figure S6). With further increases in the amount of pyrrole, however, no distinct thickness increase is found, whereas excess pyrrole polymerizes on the free SDS micelles to produce pure PPy particles. Moreover, the increase in the amount of (NH4)2S2O8 from 1 to 10 mL increases only the thickness of the PPy shell from 4.0 to 7.4 nm (Figure S7), indicating that a small amount of (NH4)2S2O8 is already enough to support pyrrole polymerization.

Figure 4. Intensity of the UV−vis absorption peak of the urchinlike Au NPs with and without the PPy shell vs the pH of the solution. Insets: TEM images of bare and PPy-coated Au NPs at different pH values. The data are recorded after the pH is altered for 1 h.

pH range of 7.0 to 9.0. The NPs aggregate in the acidic range and decompose in the basic range. In comparison, PPy-coated NPs are quite stable in the acidic range. The acidic stability is attributed to the positive PPy shell, which is fully ionized in acidic media and therewith prevents the contact of Au NPs with

Figure 3. (a) TEM image of PPy-coated urchinlike Au NPs after 1 year of storage in the aqueous solution at room temperature. (b) UV−vis spectra of PPy-coated urchinlike Au NPs after 0, 1, 6, 9, and 12 months of storage. D

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

acidic surroundings. In addition, under 808 nm NIR irradiation, the PPy-coated urchinlike Au NPs also exhibit good structural stability. The heat generated from the photothermal effect does not destroy the branched structures or the absorption spectra (Figure 5), whereas the bare NPs gradually melt into the bulk

Figure 5. (a) TEM image of PPy-coated urchinlike Au NPs after 3 h of irradiation with a 3 W/cm2 808 nm NIR laser. (b) UV−vis spectra of PPy-coated urchinlike Au NPs after 0, 1, 2, and 3 h of irradiation.

Au, leading to decreased absorbance (Figure S9). A clear comparison of the thermal stability of PPy-coated urchinlike Au NPs and NPs without a PPy shell is indicated in Figure 6. The sustained heating at 50 °C has no effect on the absorption spectra of PPy-coated NPs, revealing the good thermal stability (Figure 6a). In comparison, a gradual intensity decrease and red shift of the absorption spectra of the NPs without a PPy shell are observed (Figure 6b). Serious aggregates are also found after 6 h of heating. The photothermal and thermal studies indicate that the surface atoms of bare NPs are very active, making the urchinlike Au structures easier to melt and/or agglomerate into the bulk. The formation of the PPy shell can isolate neighboring Au NPs and therewith enhance their stability. In addition, we do not exclude the contribution of SDS to the stability enhancement. Through surface adsorption, SDS also improves the colloidal stability of the Au and/or composite NPs (Figure S10). Nevertheless, the improved pH and photothermal stability are mainly attributed to the PPy shell. The dramatically enhanced structural stability toward storage, pH, irradiation, and heat will soundly broaden the practical application of PPy-coated NPs in photothermal therapy. The photothermal behavior of PPy, urchinlike Au NPs, and PPy-coated urchinlike Au NPs are compared in Figure 7a. The

Figure 7. (a) Comparison of the photothermal behavior of urchinlike Au NPs, PPy, and PPy-coated urchinlike Au NPs with different PPy thicknesses. The thicknesses of the PPy shell are 3.5, 4.5, 6.0, and 15.6 nm, respectively. All solutions were irradiated with a 3 W/cm2 808 nm NIR laser for 5 min. The initial temperature is 22.0 °C. (b) Hela cell viabilities under 0.53 to 1.63 W/cm2 808 nm laser irradiation for 3 min in a 100 μL culture medium with 0.125 mg/mL PPy-coated urchinlike Au NPs after 4 h of incubation, as examined by MTT assay. The thickness of the PPy shell is 6.0 nm. Data are shown as the means ± standard error of the means, with * p < 0.05 and ** p < 0.01.

3 mL solutions that respectively contain PPy, urchinlike Au NPs, and PPy-coated urchinlike Au NPs with a fixed Au amount of 0.144 mg are irradiated with a 3 W/cm2 808 nm NIR laser. A temperature increment is found for all solutions after 5 min of irradiation, indicating that both PPy and

Figure 6. UV−vis spectra of (a) PPy-coated urchinlike Au NPs and (b) urchinlike Au NPs without a PPy shell after 0, 3, and 6 h of storage in 50 °C thermostatted water. E

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

urchinlike Au NPs contribute to the photothermal transduction. The photothermal effect of PPy is attributed to the broad absorbance in the red and NIR regions (Figure S11a), which is also the reason for selecting PPy as the shell material. After irradiation, the temperature of the Au and PPy solutions rises from 22.0 to 35.6 and 33.9 °C, respectively, confirming that the temperature increment results mainly from Au and slightly from PPy (Figure 7a). The PPy-coated urchinlike Au NPs exhibit extraordinary photothermal transduction, which also depends on the thickness of the PPy shell. As the PPy shell thickness increases from 3.5 to 15.6 nm, an obvious temperature increment from 41.3 to 47.2 °C is found. It indicates that a thin shell of PPy is positive with respect to enhancing the photothermal transduction by combining the effects of both urchinlike Au and PPy. However, further increases in the PPy thickness do not improve the photothermal effect very significantly. Note that the photothermal effect of PPy is also concentration-dependent (Figure S12a). The limited PPy dosage on the composite NPs cannot lead to such a high temperature. If we take the composite NPs with a 6.0 nm PPy shell as an example, then Au is still the major component. The quantity of PPy is only 4.0 wt % (Figure S11b). This implies that the higher photothermal transduction of composite NPs mainly relates to the interface of Au and PPy rather than the thickness of the PPy shell. It is known that when the diameter of Au NPs is larger than 50 nm, they strongly scatter light. The scattering component also increases with the NP size. In our system, the diameter of urchinlike Au NPs is between 55 and 200 nm. Thus, the scattering effect greatly decreases the efficiency of photothermal transduction. Although the photothermal effect of PPy is weaker than that of Au, the PPy shell may absorb the scattered light from the Au core to improve the efficiency in light harvesting. This secondary absorption of the NIR irradiation greatly enhances the photothermal transduction efficiency (Figure 7a). The effect of the PPy shell on the NIR absorption property of urchinlike Au NPs is further revealed by comparing the molar extinction coefficient of Au NPs before and after PPy envelopment according to eq 1.56 ε=

AVNPρNAV0 Lm0

η=

hS(T max − Tsurr) − Q dis I(1 − 10−A808)

(2)

h is heat transfer coefficient, S is the surface area of the container, and the value of hS can be obtained according to Figure S13. Tmax is the equilibrium temperature, and Tsurr is the temperature of the environment. Qdis expresses the heat dissipated from light absorbed by the container itself, which is measured to be 0.252 W using a weighing bottle containing 3 mL of pure water. I is the incident laser power, and it is 3 W/ cm2 in our experiment; A808 is the absorbance of the NPs at 808 nm. For the urchinlike Au NPs with a diameter of 120 nm, the η values are calculated to be 11.0, 18.6, 20.7, 24.0, and 27.5% with increasing PPy thicknesses of 0, 3.5, 4.5, 6.0, and 15.3 nm, respectively (Table 1). The results agree well with the measured temperature increment shown in Figure 7a. Table 1. Comparison of the Photothermal Transduction Efficiency (η) of urchinlike Au NPs before and after PPy Coating with Different PPy Shell Thicknesssa

a

thickness (nm)

0

3.5

4.5

6.0

15.3

η (%)

11.0

18.6

20.7

24.0

27.5

The diameter of the urchinlike Au NPs is 120 nm.

Because PPy-coated urchinlike Au NPs possess high structural stability and excellent photothermal behavior, they are employed in treating Hela cells. As the NIR irradiation intensity reaches 0.91 W/cm2, the viability of Hela cells in the presence of composite NPs begins to be suppressed (Figure 7b). The cell viabilities continue to decrease with the increase in irradiation intensity. When the intensity exceeds 1.35 W/ cm2, only 20% viability is obtained. In comparison, the NIR irradiation has no influence on the viability of Hela cells in the absence of composite NPs. Note that the lowered viability mainly results from the photothermal effect of composite NPs rather than the cytotoxicity. Without NIR irradiation, the viability is 100% from the incubation of the cells in the presence of 0.125 mg/mL composite NPs for 4 h (Figure 7b). Even when the cells are incubated for 24 h the viability is still 74% (Figure 8). The cytotoxicity mainly originates from the remaining SDS adsorbed on the composite NPs, whereas the PPy is almost nontoxic at the low dosage for coating Au NPs (Figure S12b). It is well known that heating cells between 41 and 47 °C leads to cell apoptosis.58,59 The irradiation of composite NPs quickly raises the temperature to greater than 41 °C (Figure 7a), thus promoting the apoptosis of Hela cells. Figure 9 exhibits the apoptosis staining of Hela cells, which permits a direct observation of the photothermal effect. After being irradiated by an NIR laser, the cells are stained with Hoechst 33342 and PI. The double-staining result clearly indicates that the irradiated Hela cells in the presence of composite NPs emit strong blue fluorescence (Figure 9b), whereas slight fluorescence is observed for the cells in the absence of NPs (Figure 9d). Hoechst 33342 is a blue fluorescent dye that can penetrate apoptotic cells and makes condensed chromatin brighter than that in normal cells. PI is a red fluorescent dye that penetrates only dead cells.60 After being doubly stained, the cells incubated in the presence of composite NPs and then exposed to NIR irradiation show blue luminescence, which is the color of Hoechst 33342. An obvious nuclear concentration and reduced nuclear volume are also found (Figure 9a,b). In comparison, the cells incubated in the

(1)

ε is the molar extinction coefficient; A is the absorbance at wavelength λ, which is 808 nm in our experiment; VNP is the average volume of the NPs; ρ is the density of the Au NPs, which is 19.32 g/cm3; NA is Avogadro’s constant; V0 is the volume of the solution; L is the path length of light, which is 1 cm in our experiment; and m0 is the mass of the Au NPs. On the basis of TEM observation, the average diameters of the urchinlike Au NPs are confirmed to be 55, 70, 90, 120, 150, and 200 nm, respectively (Figures S1 and S5). Thus, ε is calculated and indicated in Tables S2 and S3. In general, ε increases after the PPy coating. If we take the 120 nm Au NPs as an example, the ε of bare Au NPs is 3.5 × 1010 M−1 cm−1, whereas ε increases to 4.9 × 1010 M−1 cm−1 with a 6.0 nm PPy shell (Table S3). The increase in ε after the PPy coating is consistent with the aforementioned consideration in which the PPy shell leads to additional absorption of the composite NPs in the NIR region. Following the method developed by Roper et al.32 and Hu et al.,57 we calculated the photothermal transduction efficiency, η, according to eq 2. F

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

PPy shell significantly improves the structural stability of the urchinlike Au NPs toward storage, pH, heat, and NIR irradiation. Meanwhile, a thin PPy shell greatly enhances the photothermal transduction efficiency. A primary photothermal test indicates that the composite NPs strongly convert NIR irradiation into heat. As an example, a 6.0 nm PPy shell greatly increases the photothermal transduction efficiency to as high as 24.0%. The excellent structural stability and photothermal behavior of PPy-coated NPs will promote practical applications in the selective treatment of tumor cells after conjugating the NPs with specific targeting molecules. Although the current PPy shell prevents the linkage with targeting molecules, additional groups, such as carboxyl and amine, will be introduced by a copolymerization route. The size effect of the composite NPs on cellular uptake is also important if the NPs are used for cellular ablation. These issues are being investigated to improve the practicality of PPy-coated urchinlike Au NPs in photothermal therapy.

Figure 8. Cytotoxicity of PPy-coated urchinlike Au NPs in Hela cells, which is revealed by the incubation of Hela cells in 100 μL of culture medium with different concentrations of PPy-coated urchinlike Au NPs for 24 h, followed by MTT assay. The thickness of the PPy shell is 6.0 nm. Data are shown as the means ± standard error of the means, with * p < 0.05 and ** p < 0.01.

■

absence of composite NPs do not have a significantly nuclear concentration, and the nucleus occupies most of the cell volume (Figure 9c,d). In addition, single staining is also done using Hoechst (Figure S14). The cells are also luminescent blue as a result of the double-staining results. These confirm that the photothermal effect of PPy-coated urchinlike Au NPs leads to the apoptosis of Hela cells.

ASSOCIATED CONTENT

S Supporting Information *

TEM and SEM images of urchinlike Au NPs, comparison of the stability of Au NPs and PPy-coated Au NPs, the toxicity of pure PPy particles, single staining of Hela cells using Hoechst 33342, additional TEM images, UV−vis−NIR spectra, FTIR, DLS, and TGA data, the molar extinction coefficient, and the calculation of the photothermal transduction efficiency of PPy-coated Au NPs. This material is available free of charge via the Internet at http://pubs.acs.org.

■

CONCLUSIONS PPy is employed to coat urchinlike Au NPs to enhance their performance in photothermal therapy. The formation of the

Figure 9. (a, c) Bright-field and (b, d) fluorescent images of Hela cells after being irradiated with a 1.24 W/cm2 808 nm NIR laser for 10 min and being stained with Hoechst 33342 and propidium iodide. The cells are incubated (a, b) with and (c, d) without PPy-coated urchinlike Au NPs for 4 h. The volume and concentration of the NPs are 500 μL and 0.125 mg/mL, respectively. The thickness of the PPy shell is 6.0 nm. G

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

■

Article

(17) Zhang, H.; Wang, D. Y. Controlling the Growth of ChargedNanoparticle Chains through Interparticle Electrostatic Repulsion. Angew. Chem., Int. Ed. 2008, 47, 3984−3987. (18) Huang, X. H.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115−2120. (19) Chen, H. J.; Shao, L.; Lia, Q.; Wang, J. F. Gold Nanorods and Their Plasmonic Properties. Chem. Soc. Rev. 2013, 42, 2679−2724. (20) Lal, S.; Clare, S.; Halas, N. J. Nanoshell-Enabled Photothermal Cancer Therapy: Impending Clinical Impact. Acc. Chem. Res. 2008, 41, 1842−1851. (21) Loo, C.; Lowery, A.; Halas, N.; West, J.; Drezek, R. Immunotargeted Nanoshells for Integrated Cancer Imaging and Therapy. Nano Lett. 2005, 5, 709−711. (22) Liu, H. Y.; Chen, D.; Li, L. L.; Liu, T. L.; Tan, L. F.; Wu, X. L.; Tang, F. Q. Multifunctional Gold Nanoshells on Silica Nanorattles: A Platform for the Combination of Photothermal Therapy and Chemotherapy with Low Systemic Toxicity. Angew. Chem., Int. Ed. 2011, 50, 891−895. (23) Gao, L.; Fei, J. B.; Zhao, J.; Li, H.; Cui, Y.; Li, J. B. HypocrellinLoaded Gold Nanocages with High Two-Photon Efficiency for Photothermal/Photodynamic Cancer Therapy in Vitro. ACS Nano 2012, 9, 8030−8040. (24) Xia, Y. N.; Li, W. Y.; Cobley, C. M.; Chen, J. Y.; Xia, X. H.; Zhang, Q.; Yang, M. X.; Cho, E. C.; Brown, P. K. Gold Nanocages: From Synthesis to Theranostic Applications. Acc. Chem. Res. 2011, 44, 914−924. (25) Sau, T. K.; Rogach, A. L.; Dölinger, M.; Feldmann, J. One-Step High-Yield Aqueous Synthesis of Size-Tunable Multispiked Gold Nanoparticles. Small 2011, 7, 2188−2194. (26) Van de Broek, B.; Devoogdt, N.; D’Hollander, A.; Gijs, H.-L.; Jans, K.; Lagae, L.; Muyldermans, S.; Maes, G.; Borghs, G. Specific Cell Targeting with Nanobody Conjugated Branched Gold Nanoparticles for Photothermal Therapy. ACS Nano 2011, 5, 4319−4328. (27) Kumar, P. S.; Pastoriza-Santos, I.; Rodríguez-González, B.; de Abajo, F. J. G.; Liz-Marzán, L. M. High-Yield Synthesis and Optical Response of Gold Nanostars. Nanotechnology 2008, 19, 015606. (28) Nehl, C. L.; Liao, H. W.; Hafner, J. H. Optical Properties of Star-Shaped Gold Nanoparticles. Nano Lett. 2006, 6, 683−688. (29) Huang, X. Q.; Tang, S. H.; Liu, B. J.; Ren, B.; Zheng, N. F. Enhancing the Photothermal Stability of Plasmonic Metal Nanoplates by a Core-Shell Architecture. Adv. Mater. 2011, 23, 3420−3425. (30) Hong, S.; Shuford, K. L.; Park, S. Shape Transformation of Gold Nanoplates and Their Surface Plasmon Characterization: Triangular to Hexagonal Nanoplates. Chem. Mater. 2011, 23, 2011−2013. (31) Yavuz, M. S.; Cheng, Y. Y.; Chen, J. Y.; Cobley, C. M.; Zhang, Q.; Rycenga, M.; Xie, J. W.; Kim, C.; Song, K. H.; Schwartz, A. G.; Wang, L. V.; Xia, Y. N. Gold Nanocages Covered by Smart Polymers for Controlled Release with near-Infrared Light. Nat. Mater. 2009, 8, 935−939. (32) Ahn, W.; Roper, D. K. Transformed Gold Island Film Improves Light-to-Heat Transduction of Nanoparticles on Silica Capillaries. J. Phys. Chem. C 2008, 112, 12214−12218. (33) Richardson, H. H.; Carlson, M. T.; Tandler, P. J.; Hernandez, P.; Govorov, A. O. Experimental and Theoretical Studies of Light-toHeat Conversion and Collective Heating Effects in Metal Nanoparticle Solutions. Nano Lett. 2009, 9, 1139−1146. (34) Cole, J. R.; Mirin, N. A.; Knight, M. W.; Goodrich, G. P.; Halas, N. J. Photothermal Efficiencies of Nanoshells and Nanorods for Clinical Therapeutic Applications. J. Phys. Chem. C 2009, 113, 12090− 12094. (35) Carbó-Argibay, E.; Rodríguez-González, B.; Pacifico, J.; Pastoriza-Santos, I.; Pérez-Juste, J.; Liz-Marzán, L. M. Chemical Sharpening of Gold Nanorods: The Rod-to-Octahedron Transition. Angew. Chem., Int. Ed. 2007, 46, 8983−8987. (36) Xia, Y. N.; Xiong, Y.; Lim, B.; Skrabalak, S. E. Shape-Controlled Synthesis of Metal Nanocrystals: Simple Chemistry Meets Complex Physics? Angew. Chem., Int. Ed. 2009, 48, 60−103.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected], hcsun@ mail.jlu.edu.cn. Fax: +86 431 85193423. Tel: +86 431 85159205. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the 973 Program of China (2012CB933802 and 2009CB939701), the NSFC (21174051, 21221063, and 91123031), and the Special Project from MOST of China.

■

REFERENCES

(1) Jain, P. K.; Huang, X. H.; El-Sayed, I. H.; El-Sayed, M. A. Noble Metals on the Nanoscale: Optical and Photothermal Properties and Some Applications in Imaging, Sensing, Biology, and Medicine. Acc. Chem. Res. 2008, 41, 1578−1586. (2) Doane, T. L.; Burda, C. The Unique Role of Nanoparticles in Nanomedicine: Imaging, Drug Delivery and Therapy. Chem. Soc. Rev. 2012, 41, 2885−2911. (3) Boisselier, E.; Astruc, D. Gold Nanoparticles in Nanomedicine: Preparations, Imaging, Diagnostics, Therapies and Toxicity. Chem. Soc. Rev. 2009, 38, 1759−1782. (4) De, M.; Ghosh, P. S.; Rotello, V. M. Applications of Nanoparticles in Biology. Adv. Mater. 2008, 20, 4225−4241. (5) Kennedy, L. C.; Bickford, L. R.; Lewinski, N. A.; Coughlin, A. J.; Hu, Y.; Day, E. S.; West, J. L.; Drezek, R. A. A New Era for Cancer Treatment: Gold-Nanoparticle-Mediated Thermal Therapies. Small 2011, 7, 169−183. (6) Huang, H.-C.; Barua, S.; Sharma, G.; Dey, S. K.; Rege, K. Inorganic Nanoparticles for Cancer Imaging and therapy. J. Controlled Release 2011, 115, 334−357. (7) Hirsch, L. R.; Stafford, R. J.; Bankson, J. A.; Sershen, S. R.; Rivera, B.; Price, R. E.; Hazle, J. D.; Halas, N. J.; West, J. L. NanoshellMediated near-Infrared Thermal Therapy of Tumors under Magnetic Resonance Guidance. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13549− 13554. (8) von Maltzahn, G.; Park, J.-H.; Agrawal, A.; Bandaru, N. K.; Das, S. K.; Sailor, M. J.; Bhatia, S. N. Computationally Guided Photothermal Tumor Therapy Using Long-Circulating Gold Nanorod Antennas. Cancer Res. 2009, 69, 3892−3900. (9) Bardhan, R.; Lal, S.; Joshi, A.; Halas, N. J. Theranostic Nanoshells: From Probe Design to Imaging and Treatment of Cancer. Acc. Chem. Res. 2011, 44, 936−946. (10) Melancon, M. P.; Zhou, M.; Li, C. Cancer Theranostics with Near-Infrared Light-Activatable Multimodal Nanoparticles. Acc. Chem. Res. 2011, 44, 947−956. (11) Vogel, A.; Venugopalan, V. Mechanisms of Pulsed Laser Ablation of Biological Tissues. Chem. Rev. 2003, 103, 577−644. (12) Ntziachristos, V.; Ripoll, J.; Wang, L. V; Weissleder, R. Looking and Listening to Light: The Evolution of Whole-Body Photonic Imaging. Nat. Biotechnol. 2005, 23, 313−320. (13) Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A. Au Nanoparticals Target Cancer. Nano Today 2007, 2, 18−29. (14) Jones, M. R.; Osberg, K. D.; Macfarlane, R. J.; Langille, M. R.; Mirkin, C. A. Templated Techniques for the Synthesis and Assembly of Plasmonic Nanostructures. Chem. Rev. 2011, 111, 3736−3827. (15) Liao, H.; Nehl, C. L.; Hafner, J. H. Biomedical Applications of Plasmon Resonant Metal Nanoparticles. Nanomedicine 2006, 1, 201− 208. (16) Lin, S.; Li, M.; Dujardin, E.; Girard, C.; Mann, S. OneDimensional Plasmon Coupling by Facile Self-Assembly of Gold Nanoparticles into Branched Chain Networks. Adv. Mater. 2005, 17, 2553−2559. H

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Effect of Sodium Dodecyl Sulfate Surfactant. Chem. Lett. 2004, 37, 858−859. (56) Zhao, Y. X.; Pan, H. C.; Lou, Y. B.; Qiu, X. F.; Zhu, J. J.; Burda, C. Plasmonic Cu2‑xS Nanocrystals: Optical and Structural Properties of Copper-Deficient Copper(I) Sulfides. J. Am. Chem. Soc. 2009, 131, 4253−4261. (57) Tian, Q. W.; Jiang, F. R.; Zou, R. J.; Liu, Q.; Chen, Z. G.; Zhu, M. F.; Yang, S. P.; Wang, J. L.; Wang, J. H.; Hu, J. Q. Hydrophilic Cu9S5 Nanocrystals: A Photothermal Agent with a 25.7% Heat Conversion Efficiency for Photothermal Ablation of Cancer Cells in Vivo. ACS Nano 2011, 5, 9761−9771. (58) Vauthier, C.; Tsapis, N.; Couvreur, P. Nanoparticles: Heating Tumors to Death? Nanomedicine 2011, 6, 99−109. (59) Cherukuri, P.; Glazer, E. S.; Curley, S. A. Targeted Hyp Erthermia Using Metal Nanoparticles. Adv. Drug Delivery Rev. 2010, 62, 339−345. (60) Zhang, H. T.; Chen, G. G.; Zhang, Z. Y.; Chun, S.; Leung, B. C. S.; Lai, P. B. S. Induction of Autophagy in Hepatocellular Carcinoma Cells by SB203580 Requires Activation of AMPK and DAPK but Not p38 MAPK. Apoptosis 2012, 17, 325−334.

(37) Li, J.; Wu, J.; Zhang, X.; Liu, Y.; Zhou, D.; Sun, H. Z.; Zhang, H.; Yang, B. Controllable Synthesis of Stable Urchin-like Gold Nanoparticles Using Hydroquinone to Tune the Reactivity of Gold Chloride. J. Phys. Chem. C 2011, 115, 3630−3637. (38) Wu, H.-Y.; Liu, M.; Huang, M. H. Direct Synthesis of Branched Gold Nanocrystals and Their Transformation into Spherical Nanoparticles. J. Phys. Chem. B 2006, 110, 19291−19294. (39) de Broek, B.; Van Frederix, F.; Bonroy, K.; Jans, H.; Jans, K.; Borghs, G.; Maes, G. Shape-Controlled Synthesis of NIR Absorbing Branched Gold Nanoparticles and Morphology Stabilization with Alkanethiols. Nanotechnology 2011, 22, 015601. (40) Fang, W. J.; Yang, J.; Gong, J. W.; Zheng, N. F. Photo- and pHTriggered Release of Anticancer Drugs from Mesoporous SilicaCoated Pd@Ag Nanoparticles. Adv. Funct. Mater. 2012, 22, 842−848. (41) Kawano, T.; Niidome, Y.; Mori, T.; Katayama, Y.; Niidome, T. PNIPAM Gel-Coated Gold Nanorods for Targeted Delivery Responding to a Near-Infrared Laser. Bioconjugate Chem. 2009, 20, 209−212. (42) Tan, L. H.; Xing, S. X.; Chen, T.; Chen, G.; Huang, X.; Zhang, H.; Chen, H. Y. Fabrication of Polymer Nanocavities with Tailored Openings. ACS Nano 2009, 3, 3469−3474. (43) Zhang, Z. J.; Wang, L. M.; Wang, J.; Jiang, X. M.; Li, X. H.; Hu, Z. J.; Ji, Y. L.; Wu, X. C.; Chen, C. Y. Mesoporous Silica-Coated Gold Nanorods as a Light-Mediated Multifunctional Theranostic Platform for Cancer Treatment. Adv. Mater. 2012, 24, 1418−1423. (44) Tang, S. H.; Huang, X. Q.; Zheng, N. F. Silica Coating Improves the Efficacy of Pd Nanosheets for Photothermal Therapy of Cancer Cells Using near Infrared Laser. Chem. Commun. 2011, 47, 3948− 3950. (45) Li, F. G.; Winnik, M. A.; Matvienkob, A.; Mandelis, A. Polypyrrole Nanoparticles As a Thermal Transducer of NIR Radiation in Hot-Melt Adhesives. J. Mater. Chem. 2007, 17, 4309−4315. (46) Fonner, J. M; Forciniti, L.; Nguyen, H.; Byrne, J. D; Kou, Y.-F.; Syeda-Nawaz, J.; Schmidt, C. E Biocompatibility Implications of Polypyrrole Synthesis Techniques. Biomed. Mater. 2008, 3, 034124. (47) Yang, K.; Xu, H.; Cheng, L.; Sun, C. Y.; Wang, J.; Liu, Z. In Vitro and in Vivo Near-Infrared Photothermal Therapy of Cancer Using Polypyrrole Organic Nanoparticles. Adv. Mater. 2012, 24, 5586−5592. (48) Zha, Z. B.; Yue, X. L.; Ren, Q. S.; Dai, Z. F. Uniform Polypyrrole Nanoparticles with High Photothermal Conversion Efficiency for Photothermal Ablation of Cancer Cells. Adv. Mater. 2013, 25, 777− 782. (49) Chen, M.; Fang, X. L.; Tang, S. H.; Zheng, N. F. Polypyrrole Nanoparticles for High-Performance in Vivo near-Infrared Photothermal Cancer Therapy. Chem. Commun. 2012, 48, 8934−8936. (50) Kim, K. S.; Cho, C. H.; Park, E. K.; Jung, M.-H.; Yoon, K.-S.; Park, H.-K. AFM-Detected Apoptotic Changes in Morphology and Biophysical Property Caused by Paclitaxel in Ishikawa and HeLa Cells. PLoS ONE 2012, 7, e3066. (51) Cheng, L.; Yang, K.; Li, Y. G.; Chen, J. H.; Wang, C.; Shao, M. W.; Lee, S.-T.; Liu, Z. Facile Preparation of Multifunctional Upconversion Nanoprobes for Multimodal Imaging and DualTargeted Photothermal Therapy. Angew. Chem., Int. Ed. 2011, 50, 7385−7390. (52) Murakami, T.; Nakatsuji, H.; Inada, M.; Matoba, Y.; Umeyama, T.; Tsujimoto, M.; Isoda, S.; Hashida, M.; Imahori, H. Photodynamic and Photothermal Effects of Semiconducting and Metallic-Enriched Single-Walled Carbon Nanotubes. J. Am. Chem. Soc. 2012, 134, 17862−17865. (53) Xing, S. X.; Tan, L. H.; Yang, M. X.; Pan, M.; Lv, Y. B.; Tang, Q. H.; Yang, Y. H.; Chen, H. Y. Highly Controlled Core/Shell Structures: Tunable Conductive Polymer Shells on Gold Nanoparticles and Nanochains. J. Mater. Chem. 2009, 19, 3286−3291. (54) Chen, T.; Yang, M. X.; Wang, X. J.; Tan, L. H.; Chen, H. Y. Controlled Assembly of Eccentrically Encapsulated Gold Nanoparticles. J. Am. Chem. Soc. 2008, 130, 11858−11859. (55) Han, Y.-G.; Kusunose, T.; Sekino, T. One-pot Preparation of Core−Shell Structure Titania/Polyaniline Hybrid Materials: The I

dx.doi.org/10.1021/la401366c | Langmuir XXXX, XXX, XXX−XXX