Coating Carbon Nanosphere with Patchy Gold for Production of Highly

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Coating Carbon Nanosphere with Patchy Gold for Production of Highly Efficient Photothermal Agent Xiaoxiao Wang, Dongwei Cao, Xuejiao Tang, Jingjing Yang, Daoyong Jiang, Mei Liu, Nongyue He, and Zhi-Fei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05550 • Publication Date (Web): 28 Jun 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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ACS Applied Materials & Interfaces

Coating Carbon Nanosphere with Patchy Gold for Production of Highly Efficient Photothermal Agent Xiaoxiao Wanga, Dongwei Caoc, Xuejiao Tanga, Jingjing Yanga, Daoyong Jianga, Mei Liu b, Nongyue Heb,*, Zhifei Wanga,*

a

School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China

b

School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096,

China c

Department of Nephrology, Affiliated Drum Tower Hospital, Medical School of Nanjing

University, Nanjing, 210008, China

* Corresponding author: Prof. Nongyue He, Email: [email protected]; Dr. Zhifei Wang, Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: Gold- or carbonbased photothermal therapy (PTT) agents have shown encouraging therapeutic effects of PTT in the near-infrared region (NIR) in many preclinical animal experiments. It is expected that gold/carbon hybrid nanomaterial will possess the combinational NIR light absorption and can achieve further improvement in photothermal conversion efficiency. In this work, we design and construct a novel PTT agent by coating carbon nanosphere with patchy gold. To synthesize this composite particle with Janus structure, a new versatile approach based on a facile adsorption-reduction method was presented. Different from the conventional fabrication procedures, the formation of patchy gold in this approach is mainly a thermodynamics-driven spontaneous process. The results show that when compared with the conventional PTT agent gold nanorod the obtained nanocomposites not only have higher photothermal conversion efficiency but also perform more thermally stable. On the basis of these outstanding photothermal effects, the in vitro and in vivo photothermal performances in a MCF-7 cells (human breast adenocarcinoma cell line) and mice were investigated separately. Additionally, to further illustrate the advantage of this asymmetric structure, their potential was explored by selective surface functionalization, taking advantage of the affinity of both patchy gold and carbon domain to different functional molecules. These results suggest that this new hybrid nanomaterial can be used as an effective PTT agent for cancer treatment in future.

KEYWORDS: Photothermal therapy, patchy gold, carbon nanospheres, Janus structure, MCF-7 cell


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1. INTRODUCTION As a potential alternative or supplement to traditional cancer therapies, photothermal therapy (PTT), which uses optical absorbing agents to “cook” cancer under the near-infrared region (NIR) light irradiation, has been attracting significant attention in recent years.1,2 Up to now, various nanomaterial-based PTT agents,3 including carbon nanomaterials (such as carbon nanotube,4,5 graphene oxide6-9), semiconducting nanoparticles (NPs) (such as copper chalcogenides,10-13 cadmium chalcogenides,14,15 bismuth selenide16), and metallic nanomaterials (such as gold and silver nanocrystal17-21 and nanorod22-24), have shown encouraging therapeutic effects of PTT in many preclinical animal experiments. Meanwhile, it was also found that most of nanomaterials reported in the literatures had relative advantages and disadvantages. For example, gold nanorod is a substantial absorber, but the plasmon resonances in gold nanorod are affected by its size and shape and are susceptible to drift.25-27 Semiconductor absorption is more stable and can be controlled with doping, but the materials are often composed of toxic metals.14-16 For carbonbased PTT agent, although it can be much more biocompatible and also has a significant absorption cross section, majority of them focus on carbon nanotube and graphene oxide, which suffer from the complicated synthesis process.28,29 Therefore, it will be of interest to develop the novel hybrid nanomaterial-based PTT agent that can combine the advantages of gold and carbon and further avoid their corresponding disadvantages simultaneously. As an alternative to carbon nanotube/graphene oxide, carbon nanospheres (CNSs), which can be easily synthesized by the hydrothermal carbonization of glucose in large scale, are going to be used as carbon-based PTT agent in this work. To further enhance the absorption of NIR light and facilitate the functionality of particles, thin gold patch is further deposited on a partial surface of CNSs to form Janus structure. In comparison with gold nanorod alone, the thermal stability of


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this thin gold patch is expected to be higher due to the protection of carbon. It is worthwhile to point out that several gold/carbon hybrid nanomaterials with core-shell structure,19,24 such as golden carbon nanotube24 and carbon-coated gold nanocrystals,19 have already been reported as PTT agents. However, there is an inherent limitation associated with core-shell structure when using them as light absorbers. For a core-shell structure, the outer shell could shield the inner core from the irradiation of NIR, and thus decrease NIR absorption coefficient, which is supposed to be higher due to their combinational light absorption. When gold/carbon hybrid nanomaterial exists in the form of Janus structure, this limitation could be overcome due to the fact that both patchy gold and carbon exposed on the surface could absorb NIR light simultaneously, resulting in the increase in NIR absorption coefficient. In addition, different from the traditional symmetric gold nanoshell, such an asymmetrical structure could possess dual distinct surface properties, which facilitate the functionality in subsequent biomedical applications. This is why we try to design and construct a novel photothermal agent with Janus structure. Many efforts have been devoted to the fabrication of complex patchy and multi region Janus particles since the term Janus particle was mentioned by Pierre-Gilles de Gennes in his 1991 Nobel lecture.30,31 Several techniques, including lay-by-lay self-assembly, photo-polymerization of precursors in a microfluidic channel, the surface coating by deposition of evaporated patchforming substance onto the host substrate, have been consequently developed.30,31 However, these fabrication strategies always suffer from different limitations. For example, as for the patches obtained by the surface coating, their geometry is somewhat affected by the orientation of packed host particles as well as the angle of evaporation.32-34 In the effort to find new approaches which overcome these issues, in 2011, Taylor et al.35 reported the fabrication of


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silver patches on the surface of colloid silica particles via the facile wet-chemical method. However, as mentioned in the literature,35,36 the structure of resulting particles is instable. The formed silver patches on silica particles will degrade even when stored for a few hours or washed off the solution. The associated plasmonic properties will change accordingly. In addition, since there is no attraction between [AuClx(OH)4−x]− (x is related to the solution pH) and the surface of silica nanosphere, gold patches on silica nanosphere couldn’t be obtained directly by this approach.36 Therefore, to date, production of nanometer-sized Janus particles in a facile, versatile and large-scale way remains a challenge. To synthesize patchy gold-on CNSs (PG/CNSs) with high yield, herein we present a new versatile approach to create Janus structure via a facile adsorption-reduction method. Different from the conventional fabrication procedures,32-36 the formation of PG/CNSs in this approach mainly bases on a thermodynamics-driven spontaneous process, which greatly depends on the rate of the reduction of AuCl4- ions pre-adsorbed on the surface of CNS to Au (0). Only when the weak reductant such as ascorbic acid (AA) is used, can Au species eventually evolve into the cup-like patches. Meanwhile, this unique fabrication approach can also be used to produce patchy gold on other host nanoparticles’ surface such as silica. As the novel photothermal agent, the resulting PG/CNSs not only have higher photothermal conversion efficiency than the conventional gold nanorod, which can be attributed to the combinational light absorption by CNS and patchy gold, but also perform more thermally stable. On the basis of these outstanding photothermal effects, the in vitro and in vivo photothermal performances in a MCF-7 cells (human breast adenocarcinoma cell line) and mice were investigated separately. After administration, they were able to ablate tumors effectively without damaging healthy tissues. Additionally, to further illustrate the advantage of this asymmetric structure of PG/CNSs, their


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potential selective surface functionalization was investigated by taking advantage of the affinity of both patchy gold and carbon domain to different functional molecules. 2. EXPERIMENTAL SECTION 2.1 Materials 1-Ethyl-3-[3-dimethylaminopropyl]

carbodimide

hydrochloride

(EDC),

N-

hydroxysuccinimide (NHS), and Chloroauric acid (HAuCl4.3H2O) were purchased from Aldrich. Sodium hydroxide (NaOH), Glucose (C6H12O6), L-ascorbic acid (AA), Sodium borohydride (NaBH4) and hydrochloric acid (HCl, ≥37%) were purchased from Shanghai Chemical Reagent Corporation. Fluorescein-Isothiocyanate isomer I (FITC, 90%), branched polyethylenimine (MW=600 Da, 10 KDa, 25 KDa), α-mPEG-ω-COOH (MW=5 KDa), HOOC-PEG-COOH (MW=20 KDa) were purchased from Aladdin. All chemicals were used as received. All biological reagents, including thiolated DNA aptamer (DNA-SH), 1640 culture medium, fetal bovine serum (FBS), and trypsin, were purchased from Sangon Biotech. MCF-7 cells were obtained from Chinese Academy of Sciences Cells Bank. Water used in this experiment was purified by distillation of deionized water. 2.2 Preparation of CNSs CNSs were synthesized according to the reported method37, and further dispersed in water (0.1 mg·mL-1). 2.3 Surface modification of CNSs with PEI


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To 10 mL of CNSs aqueous solution, 2 mL of 30% (wt) PEI (MW=600 Da, 10 KDa or 25 KDa)/water was added. After 12 h reaction at 37 °C, the resulting CNSs were washed to remove free PEI, and then dispersed in 10 mL of water. 2.4 Preparation of PG/CNSs PG/CNSs were prepared via the adsorption-reduction method. Firstly, to 2 mL of HAuCl4 (1%) solution, 5 mL of PEI-modified CNSs suspension was added. After adsorption for 0.5 h, the resulting CNSs were washed, and then dispersed in 5 mL of water. The reduction of AuCl4- ions on CNSs was performed by adding 2 mL of AA aqueous solution (0.1 M) to the above solution. Then, the reaction was allowed to proceed for 2 h at room temperature. For the reduction reaction of AuCl4- ions by NaBH4, 1 mL of NaBH4 aqueous solution (0.01 M) was used instead. After washing, the obtained product was dispersed in aqueous solution with the concentration of 1 mg·mL-1. The mass fraction of Au in PG/CNSs is 75.2% determined by using inductively coupled plasma atomic emission spectroscopy (ICP-AES). To improve the dispersion stability of PG/CNSs under physiological condition, we further modified their surfaces with α-mPEG-ω-COOH (MW=5 KDa) group according to the literature38. 2.5 Temperature elevation tests 0.1 mL of PG/CNSs aqueous solution (1 mg·L-1) was serially diluted in 4 mL of water and irradiated using a 808 nm laser (diode laser, MW-GX-808/1~5000 mW, Chinese) on 0.35 cm of spot diameters at different power densities (1, 2.5, or 5 W·cm-2). During the NIR irradiation, the temperature of the solution was measured with a thermocouple linked to temperature controller


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(FLUKE) at every 30 s for total 8 min. For other samples, CNSs or Au nanorods aqueous solution was used instead. Three sets per each sample were measured. The thermal stability of PG/CNSs was tested by heating them in aqueous solution at 100 °C for various time according to the literature.25-27 2.6 Cytotoxicity Assays In vitro cytotoxicity of PG/CNSs-PEG to MCF-7 cells were measured using a MTT colorimetric assay according to the literature38. 2.7 In vitro photo-thermal therapy of PG/CNSs-PEG In vitro photo-thermal therapy of PG/CNSs-PEG was carried out according to the literature39 with the slight modification. Firstly, MCF-7 cells were seeded in a 24-well flat culture plate. After incubation overnight to allow cell attachment, the cells were then incubated with 50 µL of 1 mg·mL-1 PG/CNSs-PEG. After 24 h incubation, free PG/CNSs-PEG was removed. Next, Cells were irradiated with an 808 nm laser (2.5 W·cm-2) for 1, 2 or 4 min, respectively. Finally, Calcein-AM/propidium iodide test was employed to assess the cell viability. As the control, Cells cultured without the addition of PG/CNSs-PEG were used. 2.8 In vivo photo-thermal therapy For in vivo study, the breast tumor-bearing mice (with body weights of 18~20 g) treated with intratumorally injecting saline or PG/CNSs-PEG (50 µL, 4 mg·mL-1) were exposed to 808 nm laser (2.5 W·cm-2), and thermal images were taken every minute using an Infrared camera of


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FLUKE. Tumor size was compared at 1 day interval for up to 7 days, after which the mice were sacrificed. 2.9 Cell imaging MCF-7 cells were cultured in confocal dishes at a concentration of 1 × 104 cell/dish at 37 °C under 5% CO2 for 24 h. Then, the cells were incubated with PG/CNSs-FITC, MUC1 aptamerPG/CNSs or MUC1 aptamer-PG/CNSs-FITC for 1 h respectively. Afterward, cells were washed with PBS for three times to remove the nonspecifically adsorbed particles. Cell imaging was performed using a laser-scanning confocal fluorescent microscope (Leica, TCS-SP8). FITC was excited at 488 nm. 2.11 Characterization The morphology and size of the as-synthesized NPs were characterized by transmission electron microscopy (TEM, a JEOL JEM 2100 Field Emission Electron Microscope operated at 200 kV) and scanning electron microscopy (SEM, a Zeiss Ultra Plus field emission scanning electron microscope). The surface composition of PG/CNSs pre-adsorbed with AuCl4- ions was detected by the X-ray photoelectron spectra (XPS) recorded on an ESCALAB MK II, using a non-monochromatized Mg Kɑ X-ray as the excitation source and choosing C (1s) (284.6 eV) as the reference line. UV-vis-NIR absorbance spectrum of the as-synthesized NPs was measured on a Helios Gamma spectrophotometer. ξ potential of PG/CNSs before and after surface modification was measured using a Zetasizer NanoS. FT-IR Spectrum was collected on a Nicolet 5700 spectrometer. 3. RESULTS AND DISCUSSION


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3.1 Synthesis of PG/CNSs Herein, we presented a new and versatile route to fabricate PG/CNS by the facile adsorptionreduction method. A schematic representation of this method is summarized in Scheme 1. As shown in Scheme 1, CNSs were synthesized by a hydrothermal carbonization of glucose under 170 °C for 4 h.37 The TEM image shows that the average size of resulting CNSs is about 130 nm (Figure 1a and Figure S1 in SI). According to FT-IR analysis, both −OH and C=O groups can be found on their surfaces (the blue curve in Figure S2 in SI). In order to provide the coordination environment for the subsequent adsorption of AuCl4- ions, branched polyethylenimine (PEI) composed of lots of amine groups was then grafted on the surfaces of CNSs through the formation of imine bond between amino group from PEI and carbonyl group from CNS. In comparison with the blue curve in Figure S2, after functionalization with PEI, three new IR

Scheme 1. Schematic diagram of the preparation process of patchy gold-on CNS via an adsorption-reduction method.


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absorption peaks were observed (the red curve in Figure S2):40 the bands at 2932 and 2850 cm-1 due to the CH2 asymmetric and symmetric stretching modes of the PEI chain, the band at around 1000-1200 cm-1 due to C-N stretching vibration, and the band at 1612 cm-1 due to stretching vibration of C=N groups. Meanwhile, the previous band at 1708 cm-1 due to the stretching vibration mode of C=O groups disappears. All these new peaks demonstrate that the surfaces of CNSs have been successfully modified with PEI. The adsorption of AuCl4- ions to the surfaces of CNSs was conducted by directly mixing PEI-modified CNSs with the aqueous solution of HAuCl4. After the removal of free AuCl4- ions by the centrifugation, the resulting particles were further reduced with the weak reductant AA. In the last step, we found that the reducing capability of reductant adopted in the reaction would strongly influence the morphology of the

Figure 1. TEM images of CNS (a) and patchy gold-on CNS (PG/CNS) (b, c); HR-TEM image of PG/CNS (d); SEM image of PG/CNS (e); EDX pattern of PG/CNS (f).


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products and thereby their optical properties. If the strong reductant NaBH4 was used instead, only CNSs with their surfaces highly dispersed with Au nanoclusters were obtained. 3.2 Characterization of PG/CNSs The formation of PG/CNSs was characterized and analyzed by TEM, SEM, and Energy dispersive X-ray spectroscopy (EDX) measurements. As shown in Figure 1b and 1c, in comparison with the even surfaces of CNSs (Figure 1a), it is apparently observed that for most of CNSs, after the reduction with AA, there are one or two cup-like patches of gold with an average length of about 100 nm on their surfaces. The dark black part in the TEM image is Au because Au can adsorb more electrons in TEM due to its larger atomic number. The low magnification TEM imaging suggests that PG/CNSs-to-all particles ratio is above 98%. The corresponding HRTEM image of one representative particle in Figure 1d further confirms the structural feature of PG/CNS in which the fringes (0.238 nm) corresponding to the (004) planes of fcc Au are found on the attached particle’s surface.41 The Janus feature of the resulting particle is also verified by SEM imaging (Figure 1e), where the considerable charge contrast between spatially wellseparated Au (bright due to the high conductivity of Au) and carbon parts (dark) can be observed. From EDX shown in Figure 1f, both Au and C are present in the product in addition to the Cu substrate. So it is clear that we have successfully fabricated patchy gold on CNS by the facile adsorption-reduction method. Meanwhile, it can also be found that if the strong reductant NaBH4 was used instead, the surface of resulting CNS was highly coated by Au nanoclusters with the size of 2-3 nm (Figure 2a and 2b), indicating that the rate of reduction of AuCl4- ions to Au atom is crucial for the formation of PG/CNS.


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Along with the formation of PG/CNSs, their UV-vis-NIR extinction properties were found changed accordingly. As shown in Figure 2c, for CNSs, similar to the absorption of graphene oxide,6-8 they also exhibited an optical absorption in the visible and infrared region, which indicates that CNSs can absorb NIR light and convert it into heat. Upon the formation of the outer patchy gold, there is a significant increase in the absorption in the near-infrared region between 600 and 1100 nm, and the plasmon peak is centered near 850 nm. This feature is typical for thin patchy gold. It is well-known that gold nanomaterial exhibits distinctly defined surface plasmon resonance (SPR).27 This resonance will break into two absorption bands, one

Figure 2. Large-scale (a) and enlarged (b) TEM images of the product CNS@Au nanoclusters obtained by using NaBH4 as the reductant; the UV-vis-NIR absorbance spectra of aqueous solution containing CNSs, PG/CNSs, or CNS@Au nanoclusters (c), and the corresponding photograph of vials (d).


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corresponding to the short axis, or transverse mode (TSPR), and another to the long axis, or longitudinal mode (LSPR). The latter has lower energy or redder absorption than the former. Therefore, with the formation of thin cup-like patches of gold, the absorption band will red-shift as the plasmon oscillation decreases in energy. Meanwhile, if the strong reductant NaBH4 was used instead, only the SPR absorption band at about 550 nm similar to that of the original Au NPs in aqueous suspension was observed, further indicating that the surface of resulting CNS was just coated by the separate Au nanoclusters, which do not closely pack on the surface. Figure 2d gives the color of their aqueous solutions (CNSs: brown, PG/CNSs: blue, CNS coated with Au nanoclusters (CNS@Au nanoclusters): red). These data are well consistent with the results obtained by TEM and SEM characterization. 3.3 Growth mechanism of PG/CNSs To understand the growth mechanism of PG/CNSs, we further studied the structure and composition of resulting CNSs before the occurrence of reduction reaction. From Figure 3a, it can be found that in comparison with the grey surfaces of CNSs the resulting CNSs look darker due to the adsorption of AuCl4- ions. Figure 3b-3f illustrate the XPS spectra of the resulting CNSs. The main elements in the product are C, O, N, Cl, and Au (Figure 3b). The peak of C1s located at 284.8 eV (Figure 3c) can be attributed to C-C or C=C in CNSs. The N1s signal (Figure 3d) located at 399.9 eV further reveals the existence of PEI on CNS. The Cl 2p signal is evidenced at 197.7 eV (Figure 3e), confirming the existence of AuCl4- ions. The peaks of Au 4f are given in Figure 3f, and we can find that it consists of peaks at 89.9, 83.6, and 86.4 eV. The peak at 86.4 eV can be further resolved into the two peaks at 86.3 and 87.2 eV. In comparison with the standard binding energy (Au 4f7/2, Au 4f5/2),42 it can be concluded that these peaks can be assigned to two species: Au0 (87.2, 83.6 eV) and Au3+ (89.9, 86.3 eV), respectively.


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Figure 3. TEM image of CNS with adsorption of AuCl4- ions (a); XPS spectra of CNS with adsorption of AuCl4- ions: XPS survey spectrum (b); high resolution XPS spectra of C1s (c), N1s (d), Cl 2p (e) and Au 4f (f).


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According to the fitted results, it can also be found that the peak area of Au3+ 4f7/2 (156100) is larger than that of Au0 4f7/2 (58225), which means that the amount of Au3+species adsorbed on the surface of PEI-coated CNS is higher than that of Au0 species. As discussed above, there are some residual groups such as –OH on the surfaces of CNSs. And these groups can act as the weak reductant to partially transform AuCl4- ions to Au0 species during the adsorption of AuCl4ions to the surface of PEI-modified CNSs. All these results taken together offer clear evidence that before the occurrence of reduction reaction Au species are highly and homogeneously dispersed on the surface of PEI-modified CNSs and exist in the form of both Au0 and AuCl4ions, which lays the foundation for subsequent formation of cup-like patches of gold. Meanwhile, it was interestingly found that the final morphology of Au nanomaterials formed on the surfaces of CNSs greatly depends on the reducing capability of reductant used in the reaction. As shown above, if NaBH4 was used, which is the strong reductant and can rapidly reduce AuCl4- ions to Au0, highly dispersed Au nanoclusters were formed on CNSs. When the weak reductant AA was used instead, Au species eventually evolved into the cup-like patches after the reaction, indicating the occurrence of the dissolution of Au species pre-adsorbed and the re-deposition of the dissolved Au species on the surfaces of CNSs during the reaction. As we know, the reduction of AuCl4- ions occurs due to transfer of electrons from the reductant to AuCl4- ion, resulting in the formation of Au0. The resulting metallic gold then undergoes nucleation and growth to form Au nanomaterial, which is a thermodynamics-driven spontaneous process. In general, the more Au atoms are exposed on the exterior surface of Au nanomaterial, the less Au nanomaterial is stable. In comparison with highly dispersed Au nanoclusters formed by NaBH4 reduction, the cup-like gold patches formed by AA reduction are more energetically, resulting from the fact that the amount of Au atoms exposed on the surface of gold patches is less


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than that from Au nanoclusters formed by NaBH4 reduction. On the other hand, it is well known that for the deposition of metal atom on the substrate there are generally three growth modes (Figure 4):43-45 Frank-van der Merwe, Volmer-Weber and Stranski–Krastanov, which critically depend on strain and the chemical potential of the deposited film. In our system, the strong interaction between Au atom and NH2 groups from PEI group on the surface of CNS ensures the growth mode of Frank-van der Merwe, leading to the formation of gold patches. In addition, why Au species pre-adsorbed on the surface of CNS can first dissolve and then re-deposit to form cup-like gold patches during AA reduction can also be explained by Ostwald ripening theory.46,47 As shown in Figure 3a, before the occurrence of reduction reaction, Au species are highly and

Figure 4. The proposed formation mechanism of PG/CNSs. homogeneously dispersed on the surface of PEI-modified CNS, which is energetically unfavorable. Therefore, pre-adsorbed Au species have a tendency to detach from CNS and condense on the surface of larger Au particle. In conclusion, the formation of gold patches is the


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thermodynamics-driven spontaneous process,48 and the kinetically slow reduction resulting from the weak reductant AA allow for the occurrence of this process, as illustrated in Figure 4. Additionally, the formation mechanism of PG/CNSs proposed above can be supported by the effect of molecular weight (MW) of PEI on the morphology of patchy gold. As expected, the amount of AuCl4- ions pre-adsorbed on the surface of CNS is related to the MW of PEI. With the increase in MW of PEI (600 Da, 10 KDa, and 25 KDa), the amount of AuCl4- ions pre-adsorbed on the surface of CNS will increase too. As shown in Figure S3 (See it in SI), when the MW of PEI increases from 600 Da to 25 KDa, the size of patchy gold apparently increase, according to the micrographs, from a quarter to a half the circumference of the particles as they appear in the two dimensional images. This is understandable that more amine groups in higher MW of PEI could provide more adsorbed AuCl4- ions to form large patchy gold. Meanwhile, due to the strong interaction between Au atom and NH2 groups from PEI group, the further growth of increased Au atoms mainly occurs along the surface of CNS, which leads to the formation of patchy gold instead of bigger three-dimension island. Their corresponding UV-vis-NIR spectra demonstrate that with the increase in MW of PEI the LSPR peak from patchy gold first moves to higher wavelength (10 KDa) and then to lower wavelength (25 KDa) due to the increase in the thickness of patchy gold (Figure S4 in SI). In addition, as an extreme condition, 1,6Diaminohexane with only two amine groups was used instead of PEI 600 Da to evaluate the effect of the amount of adsorbed AuCl4- ions on the morphology of patchy gold. It can be found that beside of a few dark Au NPs most of CNSs are blank (without attachment of any Au nanomaterial), resulting from the shortage of AuCl4- ions used in the subsequent reduction reaction. Therefore, for the role of PEI played in the formation of PG/CNS, it mainly provides the coordination environment, which is essential not only for the adsorption of AuCl4- ions, but


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also for the growth of Au in the Frank-van der Merwe mode due to the strong interaction between Au atom and amine group. In the following experiments, PEI with MW of 600 Da was adopted. Based on the above discussions, herein we present the new general procedure to fabricate patchy gold-on host particle with Janus structure. Similar to the procedure shown in Scheme 1, the formation of patchy gold on host particle mainly involves three steps: (1) the surface modification of host particle with PEI group; (2) the adsorption of AuCl4- ions to the surface of host particle; (3) the control of reduction reaction kinetics of AuCl4- ions. To verify this idea, we further use silica NPs modified with PEI group (the average size is 250 nm, and the procedure for the surface modification of silica NPs with PEI groups can be found in SI) as host particles instead of PEI-modified CNSs. All other procedures are similar to those conducted in fabrication of patchy gold on CNS. As shown in Figure S5a and S5b, it can be found that after reduction by AA, several patchy golds were clearly observed on the surface of silica NPs, which can be verified by the corresponding UV-vis-NIR spectra. From Figure S5d, it can be seen that there is a significant increase in the absorption in the near-infrared region between 700 and 1100 nm due to the formation of thin patchy gold. Meanwhile, if NaBH4 was used, highly dispersed Au nanoclusters were formed on silica NP instead (Figure S5c). As mentioned in introduction, Klupp Taylor et al 36 have also reported one-pot and template-free route toproduce patchy silver on silica NPs via electrostatic force. However, for the formation of patchy gold, this strategy failed due to the lack of attraction between silica surface and AuCl4- ion. Therefore, compared with any previous methods,32-36 our approach to fabricate patchy gold seems to be more versatile and facile. 3.4 Photothermal conversion ability of PG/CNSs


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The photothermal conversion ability of obtained PG/CNSs under NIR irradiation (808 nm, continuous wave, power density: 1, 2.5, or 5 W·cm-2) was studied via an IR thermal camera, allowing measurement of temperature changes. As shown in Figure 5a, for the aqueous solution with the same concentration of CNSs and same sample volume, the aqueous solution containing CNSs increased in temperature by 3.3 °C/min and 52 °C over 8 min. The aqueous solution containing PG/CNSs induced greater temperature changes (rate =6.1 °C/min, 75 °C over 8 min). As a control, the temperature of pure water just changed from 25 to 28 °C in 8 min. The above measurements clearly demonstrate that CNS itself possesses the good photothermal conversion ability, which is similar to the light absorption of traditional graphene oxide/carbon nanotube.7,8 To the best of our knowledge, it is the first report that CNSs have been used as optical absorbing

Figure 5. Temperature elevation of the various samples as a function of irradiation time (0-8 min): (a) pure water, the aqueous dispersion of CNSs (0.1 mg·mL-1), and the aqueous dispersion of PG/CNSs (0.1 mg·mL-1); (b) the aqueous dispersion of PG/CNSs with different concentrations; (c) the NIR irradiation under the different power densities (0.1 mg·mL-1). agent. It is worthwhile to point out that different from graphene oxide/carbon nanotube CNSs can be easily synthesized by hydrothermal carbonization of glucose in large-scale. Meanwhile, the greater photothermal effect of PG/CNSs was observed, which could be attributed to the plasmonic effect from patchy gold. The effects of different concentrations of PG/CNSs and


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different power densities on the heating rate were also shown in Figure 5b and 5c, separately. As illustrated by the heating curves in Figure 5b, these solutions exhibit concentration-dependent photothermal heating effect (from 0.025 to 0.1 mg·mL-1). To demonstrate the advantage of obtained PG/CNSs over gold nanostructures used in PTT, we further compared their photothermal conversion efficiency with gold nanorods (Au NRs, length = 41−45 nm, width = 11−13 nm, Figure 6e). As shown in Figure 6a, for the aqueous solution with the same content of Au, the heating rate with PG/CNSs is greater than that with Au NRs, especially under the condition of low concentration. In addition, according to the literatures49,50, we have calculated the heat conversion efficiencies of PG/CNSs (31.6%) and Au NRs (23.1%), separately (See it in SI). So it can be concluded that PG/CNSs have higher photothermal conversion efficiency than conventional Au NRs, which can be attributed to the combinational light absorption by CNS and patchy gold. Meanwhile, the thermal stability of PG/CNSs was tested, which is important for photothermal therapy, by heating them in aqueous solution at 100 °C for various time according to the literature.25-27 After being heated for 10 h, the UV-visNIR spectrum of PG/CNS remains essentially unchanged (Figure 6b), indicating that the corresponding thermal conversion efficiency will keep constant. By carefully analyzing TEM images taken before and after being heated at 6 and 10 h, separately, no changes can be found on their Janus structure (Figure 6d). As a control, for Au NRs aqueous solution, the LSPR peak located at about 850 nm nearly disappears as the heating time is extended to 10 h (Figure 6c). As is known, the location of LSPR peak mainly depends on the aspect ratio of Au NRs. And the disappearance of LSPR peak indicates the shape transformation of Au NRs, which can be confirmed by TEM images in Figure 6e. Therefore, in comparison with the conventional PTT


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Figure 6. (a) Temperature elevation of the aqueous dispersion of Au NRs and PG/CNS with different concentrations of Au, respectively, under NIR irradiation with a power density of 5 W·cm-2 (0.4 mmol·L-1 of Au is nearly equivalent to 0.1 mg·mL-1 of PG/CNSs); Monitoring the thermal stabilities of PG/CNSs (b) and Au NRs (c) by observing the changes in their surface plasmon absorption as a function of heating time; TEM images of PG/CNSs (d) and Au NRs (e) before and after being heated at 6 and 10 h, separately.


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agent Au NRs, PG/CNSs are expected to perform more resistant to melting under photo-induced local heating besides of higher photothermal conversion efficiency. 3.5 Surface modification of PG/CNSs To enhance the dispersion of PG/CNSs under physiological condition, we further modified their surfaces with mPEG-COOH according to the procedure shown in Scheme S1 (See it in SI). And the successful surface modification is confirmed by FT-IR spectra (Figure S8a in SI) and zeta potential changes, separately (Figure S8b in SI). As shown in Figure S8a, after reacting with mPEG-COOH under the help of EDC and NHS, the absorbance at 2932 and 2850 cm-1, which can be assigned to the C-H stretching vibration from CH2- in mPEG-COOH block, increases obviously. From Figure S8b, we can find that before the linkage with mPEG-COOH PG/CNSs show a zeta potential of +12.1 mV, indicating the existence of a lot of amine groups on their surfaces. After grafting with mPEG-COOH, the corresponding zeta potential decreased to -3.2 mV. These results collectively indicate that the surface of PG/CNS was successfully coated with a layer of mPEG-COOH. After the linkage with mPEG-COOH, the hydrodynamic diameter of resulting PG/CNS is about 184.7 nm (Figure S9 in SI). Meanwhile, in comparison with other nanomaterials, the asymmetrical structure of Janus nanoparticle also provides an opportunity to specifically tune their surface chemistry, which has been proven useful for biolabeling in the later applications. As a proof of concept, herein we further examined the individual addressability of both patchy gold and carbon surfaces. To track their selective functionalization, their surface are separately modified with the recognition group and labeling group, which can be directly visualized under a confocal laser scanning microscopy (CLSM). As shown in Scheme S2 (See it in SI), the surface of carbon domain can be first tagged


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Figure 7. Confocal laser scanning microscopy images of MCF-7 cells co-incubated with MUC1 aptamer-PG/CNSs-FITC and PG/CNSs-FITC, separately, for 1 h at 37 °C. with FITC by the reaction of amine group from PEI and the isothiocyanate group from FITC. And the selective functionalization of the Au domain can be then achieved by incubating an aqueous solution of resulting NPs with thiol modified 54-mers MUC1 aptamer, which can specifically bind to MUC1 mucin on MCF-7 cells’ surface (a tumor marker in epithelial malignancies and used in immunotherapeutic and diagnostic approaches). Excess reagents were removed by centrifugation. After 1 h incubation with MCF-7 cells, the resulting cells were further examined by CLSM. As illustrated in Figure 7, the green fluorescence signals of FITCmodified PG/CNSs can be observed on MCF-7 cells’ surface due to the strong affinity of MUC1 aptamer to MUC1 mucin on MCF -7 cells’ surface. As a control, for the sample without the attachment of MUC1 aptamer, no any fluorescence signals from FITC was observed, indicating that no any FITC-modified PG/CNSs attached to the surface of MCF-7 cells due to the lack of the recognition group MUC1 aptamer. The above results clearly support the idea that the surface


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of resulting PG/CNSs can be used for selective functionalization due to the various surface properties from both Au and carbon domain. 3.6 Toxicity evaluation

Figure 8. MTT assay of MCF-7 cells viability after incubation with PG/CNSs-PEG (0-2000 µg/mL) for 24 h. To further demonstrate the potential application of resulting PG/CNSs-PEG in PTT, we studied their cytotoxicity on MCF-7 cells by determining cellular viability using an MTT assay. In the experiment, MCF-7 cells were incubated with different concentrations of NPs for 24 h in advance. As shown in Figure 8, PG/CNSs-PEG loading has no significant influence on cell viability even at high concentration of up to 2000 µg·mL-1 (cell viability ≥ 95%), demonstrating negligible cytotoxicity of particles. Therefore, the resulting PG/CNS-PEG can be further tested in vivo. 3.7 In vitro and in vivo photothermal effect of PG/CNSs-PEG


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Figure 9. Confocal microscopic images of MCF-7 cells stained by Calcein-AM/propidium iodide after the PTT treatment: (a) Negative control; (b) incubation with PG/CNSs-PEG for 24 h. The localized photothermal effect of PG/CNSs-PEG in vitro was first investigated by CalceinAM/Propidium iodide test. In the experiment, MCF-7 cells cultured with or without PG/CNSsPEG were irradiated with 808 nm for 1, 2 or 4 min, respectively. As seen in the confocal microscopic images (Figure 9a), for the negative control, all the MCF-7 cells display green calcein fluorescence even after 4 min laser irradiation, indicating that 808 laser irradiation alone could not kill cells. However, for MCF-7 cells cultured with PG/CNSs-PEG, after 2 min laser irradiation most of cells are killed and display red fluorescence resulting from the intercalation of propidium iodide in the DNA of dead cells. When the irradiation time reaches 4 min, all MCF-7 cells were killed, which further illustrates their efficacy as the PTT agent.


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Figure 10. Photothermal images of tumor site in Laser group (injected with saline) (a) and Laser + PG/CNSs-PEG group (injected with PG/CNSs-PEG) during 3 min irradiation of 2.5 W/ cm2 NIR (b). Furthermore, we conducted the animal experiments to realize in vivo PTT of PG/CNSs-PEG according to the Animal Management rules of the Ministry of health of PRC and the guidelines for the care and use of the Southeast University Laboratory Animal Center. Eight breast cancerbearing mice were divided into two groups, with one group intratumorally injected with PG/CNSs-PEG (50 µL, 4 mg·mL-1) and the other group intratumorally injected with saline and used as the control. At 1 h after injection, an 808 nm laser with a power density of 2.5 W·cm-2 was applied to irradiate the tumors for 3 min. At the same time, an IR thermal camera was used to monitor the tumor temperature during the photothermal process. It was found that the surface temperature of the tumor on mice treated with PG/CNSs-PEG rapidly increased from 23 °C to ~ 64 °C, which is high enough to kill all kinds of cancer cells, during laser irradiation (Figure 10).


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In contrast, the mice without injection of PG/CNSs-PEG showed no apparent heating effect. There is a very limited temperature increment to 36.5 °C in the laser radiated region, which is accepted and tolerated by tissue. Figure S10 (See it in SI) further gives the photos of breast cancer-bearing mice treated with the photothermal therapy for 1, 3, 5 or 7 days. These results demonstrate that laser irradiation alone or PG/CNSs-PEG injection alone does not influence the development of tumor. At the same time, upon NIR irradiation of the PG/CNSs-PEG injected tumor, necrosis appears on the tumors within 1 d due to thermal damage. After 5 d, the tumors begin to shrink, and black scars are left on the tumor sites. These results clarify that PG/CNSsPEG are an effective PTT agent for in vivo cancer therapy. 4. CONCLUSIONS We have successfully developed the novel highly efficient PTT agent PG/CNSs via the facile adsorption-reduction method. To synthesize such composite nanoparticle with Janus structure, the new versatile approach based on the thermodynamics-driven spontaneous process has been presented. To summarize, the formation of patchy gold on host particle mainly involves three steps: (1) the surface modification of host particle with PEI group; (2) the adsorption of AuCl4ions to the surface of host particle; (3) the control of reduction reaction kinetics of AuCl4- ions. Herein, PEI mainly provides the coordination environment, which is essential not only for the adsorption of AuCl4- ions, but also for the growth of Au in the Frank-van der Merwe mode due to the strong interaction between Au atom and amine group. The results show that PG/CNSs have good biocompatibility and good dispersion stability in aqueous solution, thus facilitating its biomedical applications. In comparison with the conventional PTT agent Au NRs, PG/CNSs not only have higher photothermal conversion


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efficiency under NIR irradiation, which resulting from the combinational light absorption from CNSs and patchy gold, but also perform more thermally stable. Based on these outstanding photothermal effects, the in vitro and in vivo photothermal performances in a MCF-7 cells and mice have been investigated separately. With the injection of about 0.2 mg of the resulting particle, the local temperature of tumor rapidly increased to 64.4 °C in 3 min upon laser irradiation, which is high enough to ablate the malignant cells. Additionally, to further illustrate the advantage of the asymmetric structure of PG/CNSs, their potential was explored by selective surface functionalization, taking advantage of the affinity of both Au and carbon domain to different functional molecules. These results demonstrate that this new hybrid nanomaterial can be used as an effective PTT agent for cancer treatment in future.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] NOTES The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This research is financially supported by the State key Basic Research Program of the PRC (2014CB744501), Jiangsu province natural science foundation (BK20141332), Jiangsu provincial financial support of fundamental conditions and science and technology for people's livelihood for Jiangsu key laboratory of advanced metallic materials (BM2007204) and the Fundamental Research Funds for the Central Universities.

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Coating Carbon Nanosphere with Patchy Gold for Production of Highly

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