Photothermal Therapy
Construction of Stable Chainlike Au Nanostructures via Silica Coating and Exploration for Potential Photothermal Therapy Zhen Yin,* Wei Zhang, Qiang Fu, Hua Yue, Wei Wei,* Pei Tang, Wenjing Li, Weizhen Li, Lili Lin, Guanghui Ma, and Ding Ma* A proverbial objective in nanomaterial science is to construct low-dimensional nanoparticle (NP) assemblies with fascinating properties, which have shown great promise in optical, electronic and biomedical applications.[1–4] Thereinto, organization of Au NPs into one-dimensional (1D) chainlike nanostructures has attracted a booming interest because of the unique plasmonic properties arising from the coupling effect of the Au NP’s surface plasmon resonance (SPR).[5,6] To date, self-assembly has been widely used to construct the chainlike Au nanostructures as one of the few practical strategies.[6,7] During this process, it is proposed that the formation of nanochains should be attributed to the thermodynamic balance between various particle interactive forces, such as van der Waals attractive force and electrostatic repulsive force.[8] However, most reported processes still need additional templates or coupling agents, such as biomolecules,[6,9] polymers,[6,10–13] or molecular linkers.[14–16] Even so, the structure of these chainlike Au assemblies is very sensitive to the variation of Au particle size and the sur-
Dr. Z. Yin State Key Laboratory of Hollow Fiber Membrane Materials and Processes Department of Chemical Engineering Tianjin Polytechnic University Tianjin 300387, China E-mail: [email protected] Dr. W. Zhang, Q. Fu, Dr. H. Yue, Dr. W. Wei, Prof. G. H. Ma National Key Laboratory of Biochemical Engineering Institute of Process Engineering Chinese Academy of Sciences Beijing 100190, China E-mail: [email protected] Dr. W. Zhang, P. Tang, W. J. Li, W. Z. Li, L. L. Lin, Prof. D. Ma Beijing National Laboratory for Molecular Sciences College of Chemistry and Molecular Engineering Peking University Beijing 100871, China E-mail: [email protected] Dr. W. Wei, Prof. G. H. Ma Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) Tianjin 300072, China DOI: 10.1002/smll.201400474 small 2014, DOI: 10.1002/smll.201400474
rounding environment (e.g., pH, temperature, surface ligands, and solvent polarity),[17] which have far precluded the chainlike Au nanostructures into practice. Thus, the main challenge involves constructing 1D Au nanochains with good controllability and stability, and exploring their intrinsic properties for new applications. Herein, we first describe a facile one-pot approach to self-organize Au NPs into 1D chainlike nanostructures and simultaneously stabilize with silica coating through a classical Stöber process. The synthetic procedure for the Au chain@ SiO2 nanostructures includes two main steps (Figure 1). First, the Au NPs via the traditional citrate method (Supporting Information, Figure S1) were dispersed into NaCl solution and ethanol mixture. Second, the solution of ammonia in ethanol and tetraethyl orthosilicate (TEOS) was introduced, respectively. The present approach is free of template, polymer or molecular linker. Moreover, it’s also applicable for the bigger Au particles (size >30 nm), which provide a highly versatile and reproducible process for the assembly of Au NPs and tuning of plasmons in a wider range (from 520 nm to 810 nm). In addition, the SiO2 coating endows long-term shelf stability, which paves the way to utilize the SPR sourced from the 1D Au nanostructures. Motivated by these advancements, we further explore the potential of the Au chain@SiO2 for photothermal therapy (PTT). Compared with the isolated Au NPs, as shown in Figure 2A,C, the UV-vis spectra of our products exhibited different distinguished peaks at ≈690 nm (for Au NPs with ≈13 nm size, termed Au13) and ca. 810 nm (for Au NPs with ≈38 nm size, termed Au38). Accordingly, the color of the NP solutions changed from wine red to brown purple (for Au13) and pink (for Au38) (seen in Figure S2, Supporting Information). These phenomena usually sourced from the 1D longitudinal plasmon coupling between Au NPs, thereby indicating the successful assembly of chainlike nanostructures. Further TEM observation provided direct evidence that each chain was sealed by a silica shell, resulting in a very high yield of Au chain@SiO2. (Figure 2B,D and Supporting Information Figure S3) Notably, compared with the naked Au nanochains (Figure 3D and Supporting Information Figure S4) with some small gaps (≈0.2–0.4 nm) between individuals, the encapsulated Au nanochains became fully close-packed/merged pattern due to the compression of the outside silica shell (see
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of chainlike structure. The consistent SPR band tuning can also be achieved with extensive chain growth via controlling the assembly time. Similar phenomena were observed during the Au38 assembly process due to the synergistic effect of NaCl and ammonia (Figure S8,S9, Supporting Figure 1. Schematic procedure used to construct 1D chainlike Au nanostructures with silica Information). Moreover, the TEM images shell. captured at different assembly stage clearly reveal the formation process from the high-resolution TEM images in Supporting Information naïve dimer/trimer to mature branched chainlike structure Figure S5). (Figure 3B–E). Hence, combined use of NaCl and ammonia Considering the high sensitivity of transverse configu- was necessary to get the stable and general self-assembly ration of Au plasmon resonance to the electronic coupling system for the citrate-Au NPs regardless of particle size and between NPs, we next utilized spectroscopic method to shape variation (Figure 3E for Au13 and Supporting Informonitor the particle-chain growth in situ. Taking assembly mation Figure S9 for Au38). with Au13 NPs for example, the addition of NaCl (0.1 mm) in In addition, we also noticed that the salt concentration ethanol/water mixture resulted in little change on the absorp- should be carefully tuned for the Au self-assembly protion spectra due to the very slow self-assembly process. Even cess. Systematic studies were carried out to investigate the after one month, most of the assemblies were only dimer or assembly behavior of Au13 NPs in the presence of different trimer,[8,18] as confirmed by UV-vis spectra and TEM image concentrations of NaCl, ranging from 0.1 to 1 mm in ethanol/ in Supporting Information Figure S6,S7. Once the ammonia water mixture. When the salt concentration was higher than solution was introduced, the plasmonic peak originally 0.2 mm, the assembly process occurred easily, which was conlocated at ≈520 nm rapidly decreased, whereas a shoulder at sistent with the work reported by Yin et al.[18] In this case, longer wavelength started to develop and finally evolved into the formed Au assemblies would precipitate in a very short a pronounced band (≈690 nm) after 120 min (Figure 3A). This time, which closed the time window for subsequent sol-gel new peak represented the longitudinal plasmon coupling of process. It was getting worse for bigger Au NPs (>30 nm). 1D nanostructure between Au NPs, indicating the assembly At higher salt concentration, these NPs tended to directly
Figure 2. UV-vis absorption spectra and TEM images of A,B) Au13 chain@SiO2 and C,D) Au38 chain@SiO2.
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small 2014, DOI: 10.1002/smll.201400474
Construction of Stable Chainlike Au Nanostructures for Potential Photothermal Therapy
Figure 3. A) UV-vis spectra evolution of the Au13 NPs solution after addition of the ammonia water during the self-assembly process. Typical TEM images of the assemblies at different stage B) 10 min; C) 40 min; D) 60 min, and E) 120 min.
aggregate into bulk rather than self-assembly, let alone the subsequent silica coating. Thus, in order to avoid the aggregation of Au chains during the silica coating process, the use of small-enough concentration of NaCl was pivotal for the successfully assembly/coating process. Based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, we can also discuss further the role of salt and ammonia in an ethanol medium during the self-assembly process. Due to the Au colloidal stability with negatively charged nanoparticles, the total interaction potential (VT) between Au NPs can be expressed as the sum of the electrostatic repulsion potential (Velec), the van der Waals attraction potential (VvdW), the dipolar interaction potential (Vdipole), and the charge-dipole interaction potential (Vcharge-dipole).[8,19] Usually, the Velec keep higher than VvdW in order to guarantee the stability of the Au colloidal solution. However, once the salt was added, the Velec was weakened due to the increase of ionic strength. Moreover, the ethanol media could further compromise the Velec but increase the Vdipole because of reduction in the dielectric constant of the solvent (Figure S10, Supporting Information).[8] In this case, the attractive forces between the Au NPs suppressed the electrostatic repulsive forces, thereby leading to coupling of neighboring particles.[8,18,20] Furthermore, the introduction of ammonia might reduce the surface charge density through altering the ionic strength and/or the dielectric constant of the surrounding medium, therefore leading the growth of 1D Au chains.[8] Thus, we proposed that the salt was beneficial to form dimers as activated short-range dipolar interaction forces, while the introduction of ammonia could tailor the attractive forces between NPs and dimer assemblies, thus leading to facilitate the end-to-end collision and then growth of the chain. Once the TEOS was introduced into the system, the silica shell on the NP surface would help to terminate the growth and prevent precipitation of the Au chains, therefore obtain Au chain@SiO2. As a promising alternative or supplement to current methods of treating cancer, PTT that uses optical absorbing agents to induce tumor ablation has attracted a considerable interest. Generally, the optical resonances should be tuned into near-infrared (NIR) region, where skin, tissues, small 2014, DOI: 10.1002/smll.201400474
and hemoglobin have a transmission window with a peak at approximately 800 nm.[21] It was noticed that as-fabricated 1D Au chain@SiO2 exhibited distinct absorption in the NIR region. Thus, the possibility of using them for photothermal treatment against cancer exists. Prior to examining photothermal therapy effect, the biocompatibility of the 1D core–shell nanostructures was evaluated. Both Au13 chain@SiO2 and Au38 chain@SiO2 exhibited almost no cytotoxicity even with 5 mg/mL Au concentration (Figure 4A), indicating their good biocompatibility. For photothermal treatment, HepG2 cells were pre-incubated with the Au chain@SiO2. After removal the un-internalized particles, the samples were exposed to NIR laser (808 nm, 1.5 W) irradiation for 10 min. As shown in Figure 4B, the death of HepG2 cells with Au38 chain@SiO2 was greatly higher over the result with Au13 chain@SiO2. Further insight into the photothermal therapy effect was gained by the confocal laser scanning microscope (CLSM) with a Live/Dead assay (Figure 4C,D). After treatment with Au13 chain@SiO2, approximately 50% of the cells were killed in the exposure area, and most cells outside were viable. Once incubated with Au38 chain@SiO2, the signal of dead cells was dominant in the exposure area and even spread outward, again confirming the higher photothermal damage to HepG2 cells. To investigate the mechanism of the different killing effect on cancer cells, the thermal-efficiency under NIR laser irradiation was investigated (Figure 5A and Supporting Information Figure S11). As expected, the temperature increased very slight in the pure water, whereas the Au chain@SiO2 solution could be heated immediately due to the photo-induced thermal effect resulted from the SPR absorption. Particularly, the temperature in Au38 chain@SiO2 solution increased more quickly than that in Au13 chain@ SiO2 solution. Such a higher thermal-efficiency of Au38 chain@SiO2 could be attributed to its favorable absorption around the laser wavelength. On the contrary, the aberration between Au13 chain@SiO2 absorption peak and laser wavelength compromised the optical resonances. Next, we turned our attention to the Au cellular uptake, which might also relate to the killing effect. A comparative evaluation showed that, when the uptake amount of Au13
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Figure 4. A) Viability data of HepG2 cells after 48 h exposure to Au chain@SiO2. B) CCK8 assay showing the PPT effect after 10 min irradiation (808 nm, 1.5 W). C,D) Live/Dead assay displaying the respective killing effect of C) Au13 chain@SiO2 and D) Au38 chain@SiO2 at the concentration of 3 mg/mL (Au). Scale bars: 100 µm.
chain@SiO2 was set as 100%, the value in Au38 chain@SiO2 could jump to 260%, indicating a much higher uptake amount. Similar results were obtained by CLSM (Figure 5C,D). Compared with the Au13 chain@SiO2, the amount of bright dots in the cytoplasm was significantly increased after Au38 chain@SiO2 treatment. In addition to aforementioned higher thermal-efficiency, the improved tumor cellular uptake also contributed a lot to the better killing effect against HepG2 cells. Currently, Au nanorod, nanoshell and nanocage were regarded as three major types of NIR-absorbing agent that are useful in PTT. It has been recommended that PTT treatment schedule should be one hour or so each time, twice a week, and six weeks or more. So one central concern, which has not been realized yet, is the structure integrality of these nanomaterials during the course. Taking nanorods for example, we found that the absorption peak intensity significantly decreased to 30% after 5 h irradiation due to the heat-induced deconstruction. (Supporting Information Figure S12,S13) In this aspect, the patient may have to be administrated with these nanorods repeatedly to complete the course, and the potential safety risk from the undesired Au accumulation should be carefully assessed. By contrast, only a slight slip (≈10%) of absorption intensity was observed on as-constructed Au38 chain@SiO2 even after 7 h irradiation, which could be attributed to the unique core–shell structure. (Figure S14-A) The silica shell could seal Au NP during
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PPT process and protect the chainlike structure from deconstruction. (Supporting Information Figure S14-B,D) Such a favorable property will obviously reduce dosing frequency, control safety risk, improve patient compliance, and save treatment cost. In summary, we have fabricated 1D chainlike Au nanostructures with different Au size via a facile self-assembly approach. These assemblies can be simultaneously coated with silica to form stable core–shell Au chain@SiO2 via classical Stöber process. Further functional study demonstrated their superior potential application as a safe, reusable and high-performance plasmonic PTT agent against cancer. In the future, we will continue to construct the smart thermochemo nanodevices via these 1D Au chains with mesoporous silica shell for drug loading, and functionalize the shell surface with tumor-targeting moieties for tumor specificity. Furthermore, considering the possible surface-enhanced Raman scattering (SERS) property generated from the chainlike nanostructures, we will also attempt to explore their potential for biosensors by reducing the shell thickness and introducing SERS-active molecular probe.[22–25]
Experimental Section Self-Assembly of Au NPs and Silica-Coating: Au NPs were synthesized by the traditional citrate reduction method, and then
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small 2014, DOI: 10.1002/smll.201400474
Construction of Stable Chainlike Au Nanostructures for Potential Photothermal Therapy
Figure 5. A) Photothermal efficiency and B) cellular uptake amount of Au chain@SiO2 at the concentration of 3 mg/mL (Au). C,D) CLSM images showed the respective cellular internalization of C) Au13 chain@SiO2 and D) Au38 chain@SiO2. Scale bars: 5 µm.
transferred into the NaCl aqueous solution. After the introduction of ethanol, the mixture solution was allowed to keep still for 30–90 min. Subsequently, a solution of ammonia in ethanol was added quickly. The self-assembly process could be observed through the color change clearly. In the case of the silica-coating process, a TEOS solution was introduced immediately after ammonia was added under vigorous magnetic stirring. The desired thickness of the silica shell could be obtained by adjusting the TEOS addition amount. The reaction mixtures were then stirred for another 10–12 h. Photothermal Therapy of Au Chain@SiO2: Firstly, HepG2 Cells were seeded and incubated with Au chain@SiO2. After washing with cell culture solution, cells were illuminated with an 808 nm laser. The cell survival was determined by CCK8 assay and normalized to the blank control (without nanoparticle treatment). For LSCM imaging, cells were seeded in petri dish for further illumination, and a LIVE/DEAD Cell Viability Kit was applied. Images of cell viability were acquired by a spinning-disk UltraVIEW VoX confocal system (Perkin-Elmer).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. small 2014, DOI: 10.1002/smll.201400474
Acknowledgements This work was supported by the National Natural Science Foundation of China (21303119, 81302704), and High School Science & Technology Fund Planning Project of Tianjin (20120513).
[1] Y. N. Xia, P. D. Yang, Y. G. Sun, Y. Y. Wu, B. Mayers, B. Gates, Y. D. Yin, F. Kim, Y. Q. Yan, Adv. Mater. 2003, 15, 353. [2] H. Wang, L. Chen, Y. Feng, H. Chen, Acc. Chem. Res. 2013, 46, 1636. [3] H. Kitching, M. J. Shiers, A. J. Kenyon, I. P. Parkin, J. Mater. Chem. A 2013, 1, 6985. [4] L. G. Xu, W. Ma, L. B. Wang, C. L. Xu, H. Kuang, N. A. Kotov, Chem. Soc. Rev. 2013, 42, 3114. [5] S. Lin, M. Li, E. Dujardin, C. Girard, S. Mann, Adv. Mater. 2005, 17, 2553. [6] Y. Ofir, B. Samanta, V. M. Rotello, Chem. Soc. Rev. 2008, 37, 1814. [7] S. Mann, Nat. Mater. 2009, 8, 781. [8] H. Zhang, D. Y. Wang, Angew. Chem., Int. Ed. 2008, 47, 3984. [9] A. Kuzyk, R. Schreiber, Z. Y. Fan, G. Pardatscher, E. M. Roller, A. Hogele, F. C. Simmel, A. O. Govorov, T. Liedl, Nature 2012, 483, 311. [10] H. B. Xia, G. Su, D. Y. Wang, Angew. Chem., Int. Ed. 2013, 52, 3726. [11] H. Wang, L. Y. Chen, X. S. Shen, L. F. Zhu, J. T. He, H. Y. Chen, Angew. Chem., Int. Ed. 2012, 51, 8021. [12] S. C. Biradar, M. G. Kulkarni, RSC Adv. 2013, 3, 4261.
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[13] R. Fernandes, M. Li, E. Dujardin, S. Mann, A. G. Kanaras, Chem. Commun. 2010, 46, 7602. [14] A. Gangula, J. Chelli, S. Bukka, V. Poonthiyil, R. Podila, R. Kannan, A. M. Rao, J. Mater. Chem. 2012, 22, 22866. [15] M. V. Walter, N. Cheval, O. Liszka, M. Malkoch, A. Fahmi, Langmuir 2012, 28, 5947. [16] A. Abbas, R. Kattumenu, L. M. Tian, S. Singamaneni, Langmuir 2013, 29, 56. [17] M. Li, S. Johnson, H. Guo, E. Dujardin, S. Mann, Adv. Funct. Mater. 2011, 21, 851. [18] X. G. Han, J. Goebl, Z. D. Lu, Y. D. Yin, Langmuir 2011, 27, 5282. [19] Y. D. Liu, X. G. Han, L. He, Y. D. Yin, Angew. Chem., Int. Ed. 2012, 51, 6373. [20] Y. Z. Liu, X. M. Lin, Y. G. Sun, T. Rajh, J. Am. Chem. Soc. 2013, 135, 3764.
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[21] X. H. Huang, I. H. El-Sayed, W. Qian, M. A. El-Sayed, J. Am. Chem. Soc. 2006, 128, 2115. [22] Y. J. Wong, L. F. Zhu, W. S. Teo, Y. W. Tan, Y. H. Yang, C. Wang, H. Y. Chen, J. Am. Chem. Soc. 2011, 133, 11422. [23] Y. X. Hu, Q. Zhang, J. Goebl, T. R. Zhang, Y. D. Yin, Phys. Chem. Chem. Phys. 2010, 12, 11836. [24] T. P. Tyler, A. I. Henry, R. P. Van Duyne, M. C. Hersam, J. Phys. Chem. Lett. 2011, 2, 218. [25] K. L. Wustholz, A. I. Henry, J. M. McMahon, R. G. Freeman, N. Valley, M. E. Piotti, M. J. Natan, G. C. Schatz, R. P. Van Duyne, J. Am. Chem. Soc. 2010, 132, 10903.
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Received: February 20, 2014 Revised: April 10, 2014 Published online:
small 2014, DOI: 10.1002/smll.201400474
Construction of Stable Chainlike Au Nanostructures via Silica Coating and Exploration for Potential Photothermal Therapy Zhen Yin,* Wei Zhang, Qiang Fu, Hua Yue, Wei Wei,* Pei Tang, Wenjing Li, Weizhen Li, Lili Lin, Guanghui Ma, and Ding Ma* A proverbial objective in nanomaterial science is to construct low-dimensional nanoparticle (NP) assemblies with fascinating properties, which have shown great promise in optical, electronic and biomedical applications.[1–4] Thereinto, organization of Au NPs into one-dimensional (1D) chainlike nanostructures has attracted a booming interest because of the unique plasmonic properties arising from the coupling effect of the Au NP’s surface plasmon resonance (SPR).[5,6] To date, self-assembly has been widely used to construct the chainlike Au nanostructures as one of the few practical strategies.[6,7] During this process, it is proposed that the formation of nanochains should be attributed to the thermodynamic balance between various particle interactive forces, such as van der Waals attractive force and electrostatic repulsive force.[8] However, most reported processes still need additional templates or coupling agents, such as biomolecules,[6,9] polymers,[6,10–13] or molecular linkers.[14–16] Even so, the structure of these chainlike Au assemblies is very sensitive to the variation of Au particle size and the sur-
Dr. Z. Yin State Key Laboratory of Hollow Fiber Membrane Materials and Processes Department of Chemical Engineering Tianjin Polytechnic University Tianjin 300387, China E-mail: [email protected] Dr. W. Zhang, Q. Fu, Dr. H. Yue, Dr. W. Wei, Prof. G. H. Ma National Key Laboratory of Biochemical Engineering Institute of Process Engineering Chinese Academy of Sciences Beijing 100190, China E-mail: [email protected] Dr. W. Zhang, P. Tang, W. J. Li, W. Z. Li, L. L. Lin, Prof. D. Ma Beijing National Laboratory for Molecular Sciences College of Chemistry and Molecular Engineering Peking University Beijing 100871, China E-mail: [email protected] Dr. W. Wei, Prof. G. H. Ma Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) Tianjin 300072, China DOI: 10.1002/smll.201400474 small 2014, DOI: 10.1002/smll.201400474
rounding environment (e.g., pH, temperature, surface ligands, and solvent polarity),[17] which have far precluded the chainlike Au nanostructures into practice. Thus, the main challenge involves constructing 1D Au nanochains with good controllability and stability, and exploring their intrinsic properties for new applications. Herein, we first describe a facile one-pot approach to self-organize Au NPs into 1D chainlike nanostructures and simultaneously stabilize with silica coating through a classical Stöber process. The synthetic procedure for the Au chain@ SiO2 nanostructures includes two main steps (Figure 1). First, the Au NPs via the traditional citrate method (Supporting Information, Figure S1) were dispersed into NaCl solution and ethanol mixture. Second, the solution of ammonia in ethanol and tetraethyl orthosilicate (TEOS) was introduced, respectively. The present approach is free of template, polymer or molecular linker. Moreover, it’s also applicable for the bigger Au particles (size >30 nm), which provide a highly versatile and reproducible process for the assembly of Au NPs and tuning of plasmons in a wider range (from 520 nm to 810 nm). In addition, the SiO2 coating endows long-term shelf stability, which paves the way to utilize the SPR sourced from the 1D Au nanostructures. Motivated by these advancements, we further explore the potential of the Au chain@SiO2 for photothermal therapy (PTT). Compared with the isolated Au NPs, as shown in Figure 2A,C, the UV-vis spectra of our products exhibited different distinguished peaks at ≈690 nm (for Au NPs with ≈13 nm size, termed Au13) and ca. 810 nm (for Au NPs with ≈38 nm size, termed Au38). Accordingly, the color of the NP solutions changed from wine red to brown purple (for Au13) and pink (for Au38) (seen in Figure S2, Supporting Information). These phenomena usually sourced from the 1D longitudinal plasmon coupling between Au NPs, thereby indicating the successful assembly of chainlike nanostructures. Further TEM observation provided direct evidence that each chain was sealed by a silica shell, resulting in a very high yield of Au chain@SiO2. (Figure 2B,D and Supporting Information Figure S3) Notably, compared with the naked Au nanochains (Figure 3D and Supporting Information Figure S4) with some small gaps (≈0.2–0.4 nm) between individuals, the encapsulated Au nanochains became fully close-packed/merged pattern due to the compression of the outside silica shell (see
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of chainlike structure. The consistent SPR band tuning can also be achieved with extensive chain growth via controlling the assembly time. Similar phenomena were observed during the Au38 assembly process due to the synergistic effect of NaCl and ammonia (Figure S8,S9, Supporting Figure 1. Schematic procedure used to construct 1D chainlike Au nanostructures with silica Information). Moreover, the TEM images shell. captured at different assembly stage clearly reveal the formation process from the high-resolution TEM images in Supporting Information naïve dimer/trimer to mature branched chainlike structure Figure S5). (Figure 3B–E). Hence, combined use of NaCl and ammonia Considering the high sensitivity of transverse configu- was necessary to get the stable and general self-assembly ration of Au plasmon resonance to the electronic coupling system for the citrate-Au NPs regardless of particle size and between NPs, we next utilized spectroscopic method to shape variation (Figure 3E for Au13 and Supporting Informonitor the particle-chain growth in situ. Taking assembly mation Figure S9 for Au38). with Au13 NPs for example, the addition of NaCl (0.1 mm) in In addition, we also noticed that the salt concentration ethanol/water mixture resulted in little change on the absorp- should be carefully tuned for the Au self-assembly protion spectra due to the very slow self-assembly process. Even cess. Systematic studies were carried out to investigate the after one month, most of the assemblies were only dimer or assembly behavior of Au13 NPs in the presence of different trimer,[8,18] as confirmed by UV-vis spectra and TEM image concentrations of NaCl, ranging from 0.1 to 1 mm in ethanol/ in Supporting Information Figure S6,S7. Once the ammonia water mixture. When the salt concentration was higher than solution was introduced, the plasmonic peak originally 0.2 mm, the assembly process occurred easily, which was conlocated at ≈520 nm rapidly decreased, whereas a shoulder at sistent with the work reported by Yin et al.[18] In this case, longer wavelength started to develop and finally evolved into the formed Au assemblies would precipitate in a very short a pronounced band (≈690 nm) after 120 min (Figure 3A). This time, which closed the time window for subsequent sol-gel new peak represented the longitudinal plasmon coupling of process. It was getting worse for bigger Au NPs (>30 nm). 1D nanostructure between Au NPs, indicating the assembly At higher salt concentration, these NPs tended to directly
Figure 2. UV-vis absorption spectra and TEM images of A,B) Au13 chain@SiO2 and C,D) Au38 chain@SiO2.
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Construction of Stable Chainlike Au Nanostructures for Potential Photothermal Therapy
Figure 3. A) UV-vis spectra evolution of the Au13 NPs solution after addition of the ammonia water during the self-assembly process. Typical TEM images of the assemblies at different stage B) 10 min; C) 40 min; D) 60 min, and E) 120 min.
aggregate into bulk rather than self-assembly, let alone the subsequent silica coating. Thus, in order to avoid the aggregation of Au chains during the silica coating process, the use of small-enough concentration of NaCl was pivotal for the successfully assembly/coating process. Based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, we can also discuss further the role of salt and ammonia in an ethanol medium during the self-assembly process. Due to the Au colloidal stability with negatively charged nanoparticles, the total interaction potential (VT) between Au NPs can be expressed as the sum of the electrostatic repulsion potential (Velec), the van der Waals attraction potential (VvdW), the dipolar interaction potential (Vdipole), and the charge-dipole interaction potential (Vcharge-dipole).[8,19] Usually, the Velec keep higher than VvdW in order to guarantee the stability of the Au colloidal solution. However, once the salt was added, the Velec was weakened due to the increase of ionic strength. Moreover, the ethanol media could further compromise the Velec but increase the Vdipole because of reduction in the dielectric constant of the solvent (Figure S10, Supporting Information).[8] In this case, the attractive forces between the Au NPs suppressed the electrostatic repulsive forces, thereby leading to coupling of neighboring particles.[8,18,20] Furthermore, the introduction of ammonia might reduce the surface charge density through altering the ionic strength and/or the dielectric constant of the surrounding medium, therefore leading the growth of 1D Au chains.[8] Thus, we proposed that the salt was beneficial to form dimers as activated short-range dipolar interaction forces, while the introduction of ammonia could tailor the attractive forces between NPs and dimer assemblies, thus leading to facilitate the end-to-end collision and then growth of the chain. Once the TEOS was introduced into the system, the silica shell on the NP surface would help to terminate the growth and prevent precipitation of the Au chains, therefore obtain Au chain@SiO2. As a promising alternative or supplement to current methods of treating cancer, PTT that uses optical absorbing agents to induce tumor ablation has attracted a considerable interest. Generally, the optical resonances should be tuned into near-infrared (NIR) region, where skin, tissues, small 2014, DOI: 10.1002/smll.201400474
and hemoglobin have a transmission window with a peak at approximately 800 nm.[21] It was noticed that as-fabricated 1D Au chain@SiO2 exhibited distinct absorption in the NIR region. Thus, the possibility of using them for photothermal treatment against cancer exists. Prior to examining photothermal therapy effect, the biocompatibility of the 1D core–shell nanostructures was evaluated. Both Au13 chain@SiO2 and Au38 chain@SiO2 exhibited almost no cytotoxicity even with 5 mg/mL Au concentration (Figure 4A), indicating their good biocompatibility. For photothermal treatment, HepG2 cells were pre-incubated with the Au chain@SiO2. After removal the un-internalized particles, the samples were exposed to NIR laser (808 nm, 1.5 W) irradiation for 10 min. As shown in Figure 4B, the death of HepG2 cells with Au38 chain@SiO2 was greatly higher over the result with Au13 chain@SiO2. Further insight into the photothermal therapy effect was gained by the confocal laser scanning microscope (CLSM) with a Live/Dead assay (Figure 4C,D). After treatment with Au13 chain@SiO2, approximately 50% of the cells were killed in the exposure area, and most cells outside were viable. Once incubated with Au38 chain@SiO2, the signal of dead cells was dominant in the exposure area and even spread outward, again confirming the higher photothermal damage to HepG2 cells. To investigate the mechanism of the different killing effect on cancer cells, the thermal-efficiency under NIR laser irradiation was investigated (Figure 5A and Supporting Information Figure S11). As expected, the temperature increased very slight in the pure water, whereas the Au chain@SiO2 solution could be heated immediately due to the photo-induced thermal effect resulted from the SPR absorption. Particularly, the temperature in Au38 chain@SiO2 solution increased more quickly than that in Au13 chain@ SiO2 solution. Such a higher thermal-efficiency of Au38 chain@SiO2 could be attributed to its favorable absorption around the laser wavelength. On the contrary, the aberration between Au13 chain@SiO2 absorption peak and laser wavelength compromised the optical resonances. Next, we turned our attention to the Au cellular uptake, which might also relate to the killing effect. A comparative evaluation showed that, when the uptake amount of Au13
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Figure 4. A) Viability data of HepG2 cells after 48 h exposure to Au chain@SiO2. B) CCK8 assay showing the PPT effect after 10 min irradiation (808 nm, 1.5 W). C,D) Live/Dead assay displaying the respective killing effect of C) Au13 chain@SiO2 and D) Au38 chain@SiO2 at the concentration of 3 mg/mL (Au). Scale bars: 100 µm.
chain@SiO2 was set as 100%, the value in Au38 chain@SiO2 could jump to 260%, indicating a much higher uptake amount. Similar results were obtained by CLSM (Figure 5C,D). Compared with the Au13 chain@SiO2, the amount of bright dots in the cytoplasm was significantly increased after Au38 chain@SiO2 treatment. In addition to aforementioned higher thermal-efficiency, the improved tumor cellular uptake also contributed a lot to the better killing effect against HepG2 cells. Currently, Au nanorod, nanoshell and nanocage were regarded as three major types of NIR-absorbing agent that are useful in PTT. It has been recommended that PTT treatment schedule should be one hour or so each time, twice a week, and six weeks or more. So one central concern, which has not been realized yet, is the structure integrality of these nanomaterials during the course. Taking nanorods for example, we found that the absorption peak intensity significantly decreased to 30% after 5 h irradiation due to the heat-induced deconstruction. (Supporting Information Figure S12,S13) In this aspect, the patient may have to be administrated with these nanorods repeatedly to complete the course, and the potential safety risk from the undesired Au accumulation should be carefully assessed. By contrast, only a slight slip (≈10%) of absorption intensity was observed on as-constructed Au38 chain@SiO2 even after 7 h irradiation, which could be attributed to the unique core–shell structure. (Figure S14-A) The silica shell could seal Au NP during
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PPT process and protect the chainlike structure from deconstruction. (Supporting Information Figure S14-B,D) Such a favorable property will obviously reduce dosing frequency, control safety risk, improve patient compliance, and save treatment cost. In summary, we have fabricated 1D chainlike Au nanostructures with different Au size via a facile self-assembly approach. These assemblies can be simultaneously coated with silica to form stable core–shell Au chain@SiO2 via classical Stöber process. Further functional study demonstrated their superior potential application as a safe, reusable and high-performance plasmonic PTT agent against cancer. In the future, we will continue to construct the smart thermochemo nanodevices via these 1D Au chains with mesoporous silica shell for drug loading, and functionalize the shell surface with tumor-targeting moieties for tumor specificity. Furthermore, considering the possible surface-enhanced Raman scattering (SERS) property generated from the chainlike nanostructures, we will also attempt to explore their potential for biosensors by reducing the shell thickness and introducing SERS-active molecular probe.[22–25]
Experimental Section Self-Assembly of Au NPs and Silica-Coating: Au NPs were synthesized by the traditional citrate reduction method, and then
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, DOI: 10.1002/smll.201400474
Construction of Stable Chainlike Au Nanostructures for Potential Photothermal Therapy
Figure 5. A) Photothermal efficiency and B) cellular uptake amount of Au chain@SiO2 at the concentration of 3 mg/mL (Au). C,D) CLSM images showed the respective cellular internalization of C) Au13 chain@SiO2 and D) Au38 chain@SiO2. Scale bars: 5 µm.
transferred into the NaCl aqueous solution. After the introduction of ethanol, the mixture solution was allowed to keep still for 30–90 min. Subsequently, a solution of ammonia in ethanol was added quickly. The self-assembly process could be observed through the color change clearly. In the case of the silica-coating process, a TEOS solution was introduced immediately after ammonia was added under vigorous magnetic stirring. The desired thickness of the silica shell could be obtained by adjusting the TEOS addition amount. The reaction mixtures were then stirred for another 10–12 h. Photothermal Therapy of Au Chain@SiO2: Firstly, HepG2 Cells were seeded and incubated with Au chain@SiO2. After washing with cell culture solution, cells were illuminated with an 808 nm laser. The cell survival was determined by CCK8 assay and normalized to the blank control (without nanoparticle treatment). For LSCM imaging, cells were seeded in petri dish for further illumination, and a LIVE/DEAD Cell Viability Kit was applied. Images of cell viability were acquired by a spinning-disk UltraVIEW VoX confocal system (Perkin-Elmer).
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. small 2014, DOI: 10.1002/smll.201400474
Acknowledgements This work was supported by the National Natural Science Foundation of China (21303119, 81302704), and High School Science & Technology Fund Planning Project of Tianjin (20120513).
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: February 20, 2014 Revised: April 10, 2014 Published online:
small 2014, DOI: 10.1002/smll.201400474
