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Preparation of Poly(ethylene glycol)-Modified Poly(amido amine) Dendrimers Encapsulating Gold Nanoparticles and Their Heat-Generating Ability Yasuhiro Haba, Chie Kojima, Atsushi Harada, Tomoaki Ura, Hiromichi Horinaka, and Kenji Kono* Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture UniVersity, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan ReceiVed January 11, 2007. In Final Form: February 25, 2007 Loading of HAuCl4 in poly(amido amine) G4 dendrimers having poly(ethylene glycol) (PEG) grafts at all chain ends and subsequent reduction with NaBH4 yielded PEG-modified dendrimers encapsulating gold nanoparticles (Au NPs) of ca. 2 nm diameter. The Au NPs held in the dendrimers were stable in aqueous solutions and dissolved readily, even after freeze-drying. Despite their small particle size, the heat-generating ability of Au NPs held in the dendrimer was comparable to that of widely used Au NPs with ca. 11 nm diameter under visible light irradiation. The observed excellent colloidal stability, high heat-generating ability and their biocompatible surface confirm that the PEGmodified dendrimers encapsulating Au NPs are a promising tool for photothermal therapy and imaging.
Dendrimers have attracted much interest because of their unique structures and properties.1,2 Their size, structure, and surface properties are highly controllable. In addition, their interiors can encapsulate small molecules.3-5 Considering these features, dendrimers are highly attractive materials for application in the biomedical field for tasks such as drug delivery and diagnosis.6-8 Biodistribution and toxicity of dendrimers are strongly affected by surface functionalities. Moreover, modification of their surfaces with poly(ethylene glycol) (PEG) markedly decreases their toxicity and improves their circulation time, which might contribute to their accumulation at target sites, associated with angiogenesis through so-called enhanced permeation and retention effects.9 In addition, attachment of PEG might increase its ability to encapsulate drugs by enhancing hydration around the dendrimer periphery.10 Recently, metal nanoparticles have attracted much attention for use in the biomedical field because of their interesting shapedependent and size-dependent physical and chemical properties.11 Gold nanoparticles (NPs) are particularly important metal NPs12 because they strongly absorb light in the visible region of the * To whom correspondence should be addressed: Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. Tel.: +81 72 254 9330; Fax: +81 72 254 9330; E-mail:
[email protected]. (1) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. ReV. 1999, 99, 1665-1688. (2) Tomalia, D. A.; Fre´chet, J. M. J. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2719-2728. (3) Newkome, G. R.; Moorefield, C. N.; Baker, G. R.; Saunders, M. J.; Grossman, S. H. Angew. Chem., Int. Ed. 1991, 30, 1176-1178. (4) Hawker, C. J.; Wooley, K. L.; Fre´chet, J. M. J. J. Chem. Soc., Perkin Trans. 1993, 1, 1287-1297. (5) Janssen, J. F. G. A.; de Brabander-van den Berg, E. M. M.; Meijer, E. W. Science 1994, 266, 1226-1229. (6) Liu, M.; Fre´chet, J. M. J. Pharm. Sci. Technol. Today 1999, 2, 393-401. (7) Stiriba, S.-E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 13291334. (8) Boas, U.; Heegaard, P. M. H. Chem. Soc. ReV. 2004, 33, 43-63. (9) Takakura, Y.; Mahato, R. I.; Hashida, M. AdV. Drug DeliVery ReV. 1998, 32, 93-108. (10) Kojima, C.; Kono, K.; Maruyama, K.; Takagishi, T. Bioconjugate Chem. 2000, 33, 9169-9172. (11) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chem. ReV. 2005, 105, 1025-1102. (12) Sonvico, F.; Dubernet, C.; Colombo, P.; Couvreur, P. Curr. Pharm. Des. 2005, 11, 2091-2105.
spectrum13 and convert the absorbed light to heat on a picosecond time scale.14 For those reasons, Au NPs have been considered to be promising for use in photothermal therapy15 as well as imaging16 in the biomedical field. For the use of Au NPs in the biomedical field, indeed, biocompatible surfaces should be given to them to increase their stability in the body and efficacy of their accumulation at the target. Although direct conjugation with PEG has been attempted,17 the use of carrier systems for Au NPs is an alternative and efficient method for this purpose. In a previous study, we prepared poly(amido amine) (PAMAM) dendrimers having PEG at every chain end of the dendrimer and demonstrated their potential usefulness as drug carriers.10 Considering their biocompatible surface and dendrimer features such as size controllability and molecular uniformity, they are highly attractive as a carrier for Au NPs. To elucidate their feasibility as a carrier of Au NPs, we prepared PEG-modified PAMAM dendrimers encapsulating Au NPs and investigated their stability and photothermal properties in this study. Uniform Au NPs with 2-4 nm diameters are obtainable by loading of HAuCl4 in the PAMAM dendrimers of various generations and subsequent reduction with an appropriate reducing agent, such as NaBH4.18 The AuCl4- ions are first sequestered within the dendrimer and are then chemically reduced within the (13) Link, S.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 8410-8426. (14) Link, S.; El-Sayed, M. A. Int. ReV. Phys. Chem. 2000, 19, 409-453. (15) (a) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Cancer Lett. 2006, 239, 129-135. (b) Events, M.; Saini, V.; Leddon, J. L.; Kok, R. J.; Stoff-Khalili, M., Preuss, M. A.; Millican, C. L.; Perkins, G., Brown, J. M.; Bagaria, H.; Nikles, D. E.; Johnson, D. T.; Zharov, V. P.; Curiel, D. T. Nano Lett. 2006, 6, 587-591. (c) Lapotko, D. O.; Lukianova, E.; Oraevsky, A. A. Lasers Surg. Med. 2006, 38, 631-642. (16) (a) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5, 829-834. (b) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am. Chem. Soc. 2005, 218, 2115-2120. (c) Jain, P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 7238-7248. (d) Bielinska, A.; Eichman, J. D.; Lee, I.; Baker, J. R.; Balogh, L. J. Nano Res. 2002, 4, 395-403. (e) Boyer, D.; Tamarat, P.; Maali, A.; Louis, B.; Orrit, M. Science 2002, 297, 1160-1163. (17) (a) Paciotti, G. F.; Myer, L.; Weinreich, D.; Goia, D.; Pavel, N.; McLaughlin, R. E.; Tamarkin, L. Drug DeliVery 2004, 11, 169-183. (b) Takae, S.; Akiyama Y.; Otsuka, H.; Teisaku Nakamura, T.; Nagasaki, Y.; Kataoka, K. Biomacromolecules 2005, 6, 818-824. (18) Grohn, F.; Bauer, B. J.; Akpalu, Y. A.; Jackson, E. J.; Amis, E. Macromolecules 2000, 33, 6042-6050.
10.1021/la0700826 CCC: $37.00 © 2007 American Chemical Society Published on Web 04/10/2007
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Figure 1. Time-dependent UV-vis spectra of Au NPs encapsulated by PEG-modified dendrimers (A) or unmodified dendrimers (B). The spectra of NPs were measured after centrifugation at 13 000 g for 30 min at given time intervals. The spectra before the addition of reducing agent were also indicated. Scheme 1. Preparation of PEG-Modified Dendrimer Encapsulating Au Nanoparticles
dendrimer. In addition, Hedden et al. demonstrated that starlike copolymers of PAMAM dendrimers with PEG (number average molecular weight ) 5000) arms are useful as a template for the formation of Au NPs.19 Therefore, we performed reduction of HAuCl4 by that procedure using the PEG-modified PAMAM dendrimer obtained using amine-terminated PAMAM G4 dendrimers and poly(ethylene glycol) monomethyl ether with the number average molecular weight of 2000;10 it was used as the template (Scheme 1). For comparison, Au NPs were also prepared using the unmodified PAMAM G4 dendrimer or by standard citrate reduction.20 (19) Hedden, R. C.; Bauer, B. J.; Smith, A. P.; Grohn, F.; Amis, E. Polymer 2002, 43, 5473-5481. (20) (a) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (b) Kooji, E. S.; Brouwer, E. A. M.; Wormeester, H.; Poelsema, B. Langmuir 2002, 18, 7677-7682.
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Figure 2. TEM analysis of Au NPs encapsulated by PEG-modified dendrimers (A,B) or unmodified dendrimers (C,D) and Au NPs prepared by sodium citrate reduction (E,F). The size distributions estimated from TEM images, A, C, and E, were shown in panels B, D, and F, respectively.
Figure 1 shows changes in the absorption spectra of aqueous solutions of HAuCl4 and the PEG-modified or unmodified PAMAM G4 dendrimer mixed at the ratio of 55/1 (mol/mol) before and after addition of a 5-fold molar excess of NaBH4 to HAuCl4. No absorption was observed at wavelengths greater than 500 nm without NaBH4 for these HAuCl4 solutions containing dendrimers. However, when the reducing agent was added, solutions containing either dendrimer showed similarly increased absorbance around 520 nm derived from the surface plasmon resonance.21 This result indicates that Au NPs were generated in PAMAM dendrimers even when the PEG grafts are combined to its periphery. As shown in Figure 1A, the spectra of Au NP suspensions with the PEG-modified dendrimer were invariable for 5 days. However, the absorbance for a Au NP suspension with the unmodified dendrimers decreased with time and had almost entirely diminished in 3 days (Figure 1B). We observed that large aggregates were formed in the suspension of the unmodified dendrimer encapsulating Au NPs within 24 h. In addition, observation with TEM revealed that intensive association and fusion of Au NPs occurred in the suspension of unmodified dendrimers encapsulating Au NPs (see Figure S1, Supporting Information). Apparently, the PEG-modified dendrimer has a higher ability to stabilize Au NPs than the unmodified dendrimer. Probably, the steric effect of PEG chains strongly inhibits contact between Au NPs encapsulated in dendrimers and suppresses aggregation of Au NPs. (21) Daniel, M. C.; Astruc, D. Chem. ReV. 2004, 104, 293-346.
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Figure 3. UV-vis spectra of Au NPs encapsulated by PEG-modified dendrimers (a) or unmodified dendrimers (b) and Au NPs prepared by citrate reduction (c) in aqueous solution before and after freeze-drying. The spectra of NPs were measured without centrifugation.
We next characterized Au NPs generated in the dendrimer interior using transmission electron microscopy (TEM). As shown in Figure 2, Au NPs prepared with the PEG-modified dendrimer have ca. 2 nm average diameter with a narrow size distribution compared to Au NPs obtained with the unmodified dendrimers, which have ca. 3 nm average diameter with a broad size distribution. The diameters of the smallest Au NPs are approximately the same for both dendrimers. This observation suggests that the larger nanoparticles prepared with the unmodified dendrimer might be formed through aggregation of smaller particles. The Au particles generated in the unmodified dendrimer might not be effectively isolated from the outer phase and thereby tend to form aggregates with Au particles held in other dendrimer molecules. In contrast, Au particles retained in the PEG-modified dendrimer might be covered efficiently with highly hydrated PEG chains. Since the PEG chains keep Au particles from aggregating, the resulting particles exhibit smaller size and narrower size distribution. Previously, Hedden et al. reported that the use of the starlike copolymer of PAMAM G4 dendrimers having 60 PEG grafts as a template controls nanoparticle size of Au in the same manner as the unmodified dendrimer.19 However, our result demonstrates the importance of coverage with PEG chains for better control of particle size induced by the PAMAM dendrimers. Complete modification of the dendrimer chain ends with PEG chains might be important for particle size control. The Au NPs prepared with sodium citrate for reference were also observed using TEM, indicating their sharp size distribution with a mean of 11 nm. To further confirm the role of PEG chains in the PEG-modified dendrimer-mediated production of Au NPs, we prepared Au particles using free PEG (Mn ) 2000) instead of the PEG-modified dendrimer. Observation using TEM showed that larger nanoparticles with a broader size distribution were generated, indicating that PEG chains cannot act as a template for nanoparticle formation (see Figure S2, Supporting Information). The PEG chains attached to the dendrimer surface show another excellent effect of improving colloidal stability of Au NPs, as observed for the dissolution property of dried samples of Au NPs. Figure 3 shows that Au NPs encapsulated in the PEGmodified dendrimers can be readily dissolved in water after freezedrying. The resultant suspension exhibited nearly identical absorption spectra to that of Au NPs before freeze-drying (Figure 3A). In contrast, absorbance of Au NPs attributable to the surface plasmon resonance decreased markedly for the unmodified PAMAM dendrimers encapsulating Au NPs after freeze-drying, probably because significant aggregation of Au NPs occurred during freeze-drying (Figure 3B). In fact, significant precipitation was observed when the dried sample of the unmodified dendrimer containing Au NPs was dissolved in water. Indeed, bare Au NPs were unable to form their suspension with absorption based on the surface plasmon resonance because of their intensive
Figure 4. (A) Photoirradiation-induced increase in temperature of water containing Au NP-encapsulating PEG-modified dendrimer (triangles), empty PEG-modified dendrimer (diamonds), Au NP prepared by citrate reduction (squares), or pure water (circles) as a function of irradiation time. Concentrations of Au atoms in the solution were 275 µM (open symbols) and 55 µM (closed symbols). Empty dendrimer concentration was 10 µM. (B) Photoirradiationinduced increase in temperature of water containing PEG-modified PAMAM dendrimers encapsulating Au NP (triangles) and Au NP prepared by sodium citrate reduction (squares) solutions after 5 min as a function of Au atom concentration.
aggregation during freeze-drying (Figure 3C). In addition, we observed that the PEG-modified dendrimer containing Au NPs dissolved in various organic solvents such as N,N′-dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), and methanol (See Figure S3, Supporting Information). The high colloidal stability and excellent solubility of PEG-attached PAMAM dendrimer containing Au NPs should be of great importance for their practical use. Photothermal properties of Au NPs encapsulated in the PEGmodified dendrimer were compared with those of Au NPs prepared with sodium citrate, which has been used for this purpose.15a,22 Figure 4 shows the effect of light irradiation (532 nm) on the temperature of their suspensions in water. The light irradiation did not affect the temperature of water and aqueous solution of the empty PEG-modified dendrimer. However, aqueous suspensions of Au NPs either encapsulated in the PEGattached dendrimer or free increased their temperatures with (22) Jones, C. D.; Lyon, L. A. J. Am Chem. Soc. 2003, 125, 460-465.
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time under light irradiation, indicating that the Au NPs retain photothermal properties, even when encapsulated in the PEGmodified dendrimer. In addition, the extent of the light-induced temperature increase was dependent on the concentration of Au NPs. The increment of the suspension temperature after 5 min light irradiation was plotted against the Au atom concentration (Figure 4B). The extent of temperature elevation was enhanced with the concentration of Au NPs. It reached a constant level at about 0.5 mM of Au atoms, where too many Au NPs might exist in the suspension. Comparison of their photothermal properties revealed that the heat-generating ability of Au NPs encapsulated in the PEGmodified dendrimer was lower than that of conventional Au
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NPs, but the difference was slight. Therefore, the excellent photothermal properties, together with the high colloidal stability and biocompatible surface, confirm that the PEG-modified dendrimers encapsulating Au NPs are a promising tool for photothermal therapy and imaging. Efforts are being made to evaluate their biological activity and to extend their absorption spectra to the infrared region. Supporting Information Available: Additional experimental details as mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org. LA0700826
