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PNIPAM Gel-Coated Gold Nanorods for Targeted Delivery Responding to a Near-Infrared Laser Takahito Kawano,† Yasuro Niidome,† Takeshi Mori,†,‡ Yoshiki Katayama,†,‡,§ and Takuro Niidome*,†,‡,# Department of Applied Chemistry, Faculty of Engineering, and Center for Future Chemistry, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan, and CREST and PRESTO, Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan. Received November 6, 2008; Revised Manuscript Received December 22, 2008
Gold nanorods can be used as photothermal converters, permitting near-infrared (NIR) light to be transmitted deep into tissues without causing damage. We prepared hybrid nanorods with a core-shell structure, i.e., a single gold nanorod encapsulated in a poly (N-isopropylacrylamide) nanogel. Hybrid nanorods demonstrated remote, reversible, pulsatile phase transition and in vivo action after irradiation using a NIR laser.
The development of in vivo delivery of drugs using functional nanomaterials is a cornerstone of successful cancer therapy (1-4). Strategies for the accumulation and release of drugs with nanomaterials have been intensively developed, including those caused by external stimuli (e.g., temperature, light, magnetic field, ultrasound, specific molecules) (5-9). External stimuli trigger conversion of the physicochemical properties of the drug carrier, so the pharmacokinetics of the injected drug could be remotely controlled from outside the tissues in vivo. Among stimuli-responsive drug carriers, thermo-responsive polymers such as poly(N-isopropylacrylamide) (PNIPAM) induced phase transition at a lower critical solution temperature (LCST) and are widely used in biomaterial science (10, 11). Ultraviolet- and visible light-sensitive materials can be delivered and release drugs after responding to external stimuli (12). Current strategies have not enabled the accumulation and release of drugs to be controlled within a limited area without tissue damage. In this study, as a new class of external stimuli, we focused on near-infrared (NIR) light (650-900 nm). This allowed maximal penetration of light of tissues due to minimal optical absorption by intrinsic components such as hemoglobin (900 nm) (13). NIR light can be transmitted deep into tissue without significant damage and could have biomedical applications. Rod-shaped gold nanoparticles (“gold nanorods”) have an intense absorption band in the NIR region and show highly efficient photothermal conversion (14-16). The photothermal effect of gold nanorods actuated by irradiating NIR light induces phase transition of the thermo-responsive polymer. Using PNIPAM hydrogel-coated gold nanorods, NIR light can trigger conversion of the hydrophobic nanorod surface and formation of aggregates. In targeted delivery, PNIPAMcoated gold nanorods circulate freely after systemic injection; the NIR laser then irradiates to the targeted site, and nanorods form aggregates and accumulate at the irradiated site without tissue damage (17, 18). Our research group has prepared capillary PNIPAM hydrogels containing gold nanorods; irradiation by an NIR laser induced rapid shrinkage of the gels and released the incorporated drug (19). Kumacheva et al. prepared hybrid PNIPAM microgels, * Corresponding author. E-mail:
[email protected]. ac.jp; (+81) 92-802-2851. † Department of Applied Chemistry. ‡ Center for Future Chemistry. § CREST. # PRESTO.
which successfully absorbed cationic surfactant-stabilized gold nanorods on submicrometer-sized spheres of PNIPAM-coacrylic acid hydrogels by electrostatic interaction (20). Recently, this group showed that electrostatic interaction alone was not the governing force driving the absorption gold nanorods into microgels (21). However, for in vivo delivery based on systemic injection, applying the hybrid microgels would be disadvantageous because the size was too large to circulate in blood flow. PNIPAM gels loaded with gold nanorods should be nanosized and well-dispersed within the blood to enable targeted drug delivery using hybrid PNIPAM nanogels after systemic injection. Our strategy involved using hybrid nanogels with a welldefined core-shell structure, i.e., a single gold nanorod encapsulated in a PNIPAM nanogel. The core-shell structure of NIPAM gel-coated nanorods prevented aggregation and dissociation by nonspecific interaction with blood components. We developed a novel preparation of core-shell PNIPAM gelcoated gold nanorods by colloid-template polymerization. Hybrid nanogels were used to demonstrate photothermal phase transition by an NIR laser and remote actuation after systemic injection in vivo. PNIPAM gel-coated gold nanorods were prepared by colloid-template polymerization and silica etching (Figure 1). Gold nanorods had a length of 54.8 nm and an aspect ratio of 5.7. They were stabilized by the cationic detergent cetyltrimethylammonium bromide (CTAB) in aqueous solution (Figure 2A) (22-24). First, gold nanorods were modified with the amphiphilic polymer poly(ethylene glycol) (PEG) (25). PEG-modified gold nanorods were coated with a silica layer using the modified Sto¨ber method, which is based on the hydrolysis of tetraethyl orthosilicate (TEOS) in ethanol/water mixture in the presence of nanorods, which act as “seeds” (26, 27). After coating with silica shells, nanorods were dispersed without aggregation. Silica shells with a thickness of 16.7 nm around the nanorods were observed by transmission electron microscopy (Figure 2B). Spectra of the silica-coated nanorods showed a red shift of the surface plasmon band corresponding to the longitudinal oscillation mode because of an increase in local refractive index around the nanorods produced by the silica shells (Figure 2G) (28, 29). The silane-coupling agent 3-(methacryloxy)propyl triethoxysilane (MPS) was used to modify the surface of the silica-coated nanorods, which led to the formation of methacryl groups on the surface.
10.1021/bc800480k CCC: $40.75 2009 American Chemical Society Published on Web 01/09/2009
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Figure 1. Preparation of PNIPAM-coated gold nanorods (schematic).
Figure 2. (A-F) Transmission electron microscopy (TEM) images of gold nanorods. (G) UV-visible-NIR spectra of gold nanorods.
Second, silica-coated nanorods were modified with PNIPAM shells by precipitation polymerization (30, 31). In the presence of MPS-modified silica nanorods, precipitation polymerization of NIPAM and N,N′-methylene bisacrylamide (MBA) as crosslinker was done using potassium persulfate (KPS) as an initiator, resulting in the formation of PNIPAM gel/silica-coated gold nanorods. Figure 2C,D shows the core of a single silica-coated gold nanorod modified with the shell of PNIPAM gel. Successful PNIPAM modification involved polymerization at a low concentration of gold nanorod solution (final gold atom concentration of 0.5 mM): this prevented aggregation between nanorods. Thickness of the PNIPAM shells could be adjusted by changing the amount of the NIPAM monomer and concentration of the silica-coated gold nanorods. A blue shift of the longitudinal
surface plasmon band would be due to a change in the local refractive index produced by the PNIPAM shell. Silica templates were selectively etched using hydrofluoric acid. A hollow PNIPAM nanocapsule containing a single gold nanorod was observed (Figure 2E,F). Increase of absorption in the visible region (∼500 nm) was due to light scattering of PNIPAM nanogels (Figure 2G). The temperature-induced change in size of the PNIPAMcoated gold nanorods with different cross-linking densities was measured by dynamic light scattering. All nanogels exhibited thermoresponse discontinuous phase-transition behavior (Figure 3A). Nanogels with lower cross-linking density (7.5%) showed a sharper phase transition curve than those with higher crosslinking densities (10% and 12.5%). Phase transition of all nanogels occurred at the same temperature (about 34 °C). We investigated the photothermal phase transition of nanogels by irradiation using a focused NIR laser of wavelength 807 nm (Figure 3B). Here, to avoid aggregation, we employed a low concentration (50 µg/mL) of the nanogels. Small shrinkage of nanogels was observed after irradiation using an NIR laser for 10 min at low power (2.1 W/cm2). Nanogels shrank considerably and rapidly when irradiated using a laser at high power (3.4 W/cm2 and 4.2 W/cm2). A cycle of shrinkage and swelling could be induced by switching the laser on and off. We roughly calculated input energy into a gold nanorod and required energy to increase the temperature of the NIPAM layer on the gold nanorod by 20 °C. The nanogel was assumed as a sphere (φ ) 300 nm), the volume of the nanorod in the gel was insignificat, and the specific heat was the same as that of water. As a result of the calculation, the input energy into one gold nanorod (1.1 × 10-7 J) was 100-fold higher than the required energy to increase the temperature (1.2 × 10-9 J). It is enough to induce the phase transition of the PNIPAM layer, even the heat diffused into the silica layer and the surroundings. Gold nanorods did not exhibit a decrease or shift of the absorption peak due to irradiation by the NIR laser (data not shown). Dansylamide (which gives a fluorescent spectrum sensitive to hydrophobic environment) was mixed with nanogels, and then the mixture was irradiated by the NIR laser at various powers to investigate the nanogel environment. When the power of the NIR laser was raised, the fluorescence peak was blue-shifted and its intensity increased (Figure 3C). This indicated that the dye molecules
Figure 3. (A) Temperature dependence of hydrodynamic diameters of PNIPAM-coated gold nanorods with increasing cross-linking density. (B) Hydrodynamic diameters of PNIPAM-coated gold nanorods upon irradiation using a NIR laser with increasing power density. Periods of laser irradiation are indicated by the gray area. (C) Fluorescence spectra of dansylamide in the presence of PNIPAM-coated gold nanorods at several laser powers.
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Figure 4. (A) Distributions of PNIPAM-coated gold nanorods after injection (i.v.) with or without irradiation using an NIR laser. (B) White and light-gray bars indicate injection of nanogels alone after 10 and 30 min, respectively. Black bars indicate injection of nanogels followed by laser irradiation. Dark-gray bars indicate injection of PEG-modified gold nanorods and laser irradiation.
were in a more hydrophobic environment than the original environment due to phase transition of the PNIPAM gel. Next, we tried a feasible study on in vivo application, that is, accumulation of nanogels at laser-irradiated site in mice due to the hydrophobic surfaces induced by the photothermal effect. The hydrophobic surface could interact with cellular membrane or extracellular matrix. The nanogels with high concentration (2.5 mg/mL) were systemically injected into mice. The right kidney was immediately irradiated using the NIR laser (807 nm, 3.4 W/cm2) (Figure 4A). Organs were subsequently collected, and the amounts of the gold nanorods in organs were evaluated by inductively coupled plasma mass spectrometry (ICP-MS; Figure 4B). When a solution of PNIPAM-coated gold nanorods was injected, nanogels were distributed primarily in the blood (47%) and lungs (46%) after 10 and 30 min, respectively. The LCST of the nanogels was 34 °C lower than body temperature, but they circulated in the blood within 10 min because of a slow phase transition response to a jump in environmental temperature (19). After 30 min, nanogels accumulated in the lungs. This was due to formation of a hydrophobic surface on the nanogel after full phase transition at body temperature. Significant accumulation of gold in the irradiated right kidney was observed after irradiation of the right kidney for 10 min immediately after injection of nanogels. Accumulation was not observed in the left kidney, and the amount of gold in the blood was lower than in the case without laser irradiation. PNIPAM-coated gold nanorods, which circulated in the blood after injection, accumulated due to induced rapid photothermal phase transition in the irradiated site. Gold nanorods were also detected in the lungs. Hydrophobic nanogels that could not accumulate in the irradiated site would be trapped in the capillary vessels in the lungs. In the case of injection of PEG-modified gold nanorods followed by laser irradiation, gold nanorods were primarily found in the blood, similar to the case of nanogels without laser irradiation. These results suggested that accumulation of nanogels in targeted sites was due to photothermal phase transition and aggregation of PNIPAM nanogels after irradiation using a NIR laser. In summary, we prepared hybrid nanogels (a single gold nanorod encapsulated in a PNIPAM nanogel) by colloid-template polymerization and silica etching. PNIPAM-coated gold nanorods demonstrated photothermal phase transition and accumulation in local targeted sites which were irradiated. For in vivo application, hybrid nanogels should be optimized with respect to the LCST, size, and sensitivity by changing monomer composition and nanorod size to induce a sharp transition in the relevant physiologic range (38-42 °C). We used gold nanorods as a photothermal agent to absorb NIR light that can penetrate tissues. Gold nanorods are also used as probes for in vivo imaging techniques (two-photon luminescence, photoabsorption) (32, 33). NIPAM gel-coated nanorods may enable simultaneous application of targeted delivery of drugs, photothermal therapy, and bioimaging using a NIR laser.
ACKNOWLEDGMENT This research was supported by the Precursory Research for Embryonic Science and Technology (PRESTO) and the Core Research Program for Evolution Science and Technology (CREST) from the Japan Science and Technology Agency. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http:// pubs.acs.org.
LITERATURE CITED (1) Heath, J. R., and Davis, M. E. (2008) Nanotechnology and cancer. Annu. ReV. Med. 59, 251–265. (2) Haag, R., and Kratz, F. (2006) Polymer therapeutics: concepts and applications. Angwe. Chem., Int. Ed. 45, 1198–1215. (3) Peer, D., Karp, J. M., Hong, S., Farokhzad, O. C., Margalit, R., and Langer, R. (2007) Nanocarriers as an emerging platform for cancer therapy. Nat. Biotechnol. 2, 751–760. (4) Ferrari, M. (2005) Cancer nanotechnology: opportunities and challenges. Nat. ReV. Cancer 5, 161–171. (5) Bae, Y. H., Okano, T., and Kim, S. W. (1991) Replace “Onoff” thermocontrol of solute transport. I. Temperature dependence of swelling of N-isopropylacrylamide networks modified with hydrophobic components in water. Pharm. Res. 8, 531–537. (6) Nishiyama, N., Iriyama, A., Jang, W. D., Miyata, K., Itaka, K., Inoue, Y., Takahashi, H., Yanagi, Y., Tamaki, Y., Koyama, H., and Kataoka, K. (2005) A novel gene carrier enveloped with dendrimer-based photosensitizer for photochemical transfection in ViVo. Nat. Mater. 4, 934–941. (7) Derfus, A. M., Maltzahn, G., Harris, T. J., Duza, T., Vecchil, K. S., Ruoslahti, E., and Bhatia, S. N. (2007) Remotely triggered release from magnetic nanoparticles. AdV. Mater. 19, 3932–3936. (8) Kost, J., Leong, K., and Langer, R. (1989) Ultrasound-enhanced polymer degradation and release of incorporated substances. Proc. Natl. Acad. Sci. U.S.A. 86, 7663–7666. (9) Fischel-Ghodsian, F., Brown, L., Mathiowitz, E., Brandenburg, D., and Langer, R. (1988) Enzymatically controlled drug delivery. Proc. Natl. Acad. Sci. U.S.A. 85, 2403–2406. (10) Yoshida, R., Sakai, K., Okano, T., and Sakurai, Y. (1994) Modulating the phase transition temperature and thermosensitivity in N-isopropylacrylamide copolymer gels. J. Biomater. Sci. Sci. Polym. 6, 585–598. (11) Nayak, S., and Lyon, L. A. (2005) Soft Nanotechnology with Soft Nanoparticles. Angew. Chem., Int. Ed. 44, 7686–7708. (12) Suzuki, A., and Tanaka, T. (1990) Phase transition in polymer gels induced by visible light. Nature 346, 345–347. (13) Weissleder, R. (2001) A clearer vision for in ViVo imaging. Nat. Biotechnol. 19, 316–317. (14) Yu, Y.-Y., Chang, S.-S., Lee, C. -L., and Wang, C. R. C. (1997) Gold Nanorods: electrochemical synthesis and optical properties. J. Phys. Chem. B 101, 6661–6664. (15) Link, S., Mohamed, M. B., and El-Sayed, M. A. (1999) Simulation of the optical absorption spectra of gold nanorods as a function of their aspect ratio and the effect of the medium dielectric constant. J. Phys. Chem. B 103, 3073–3077.
212 Bioconjugate Chem., Vol. 20, No. 2, 2009 (16) Chou, C.-H., Chen, A.-D., and Wang, C. R. C. (2005) Highly efficient, wavelength-tunable, gold nanoparticle based optothermal nanoconvertors. J. Phys. Chem. B 109, 11135–11138. (17) Dreher, M. R., Liu, W., Michelich, C. R., Dewhirst, M. W., and Chilkoti, A. (2007) Thermal cycling enhances the accumulation of a temperature-sensitive biopolymer in solid tumors. Cancer Res. 67, 4418–4424. (18) Raucher, D., and Chilkoti, A. (2001) Enhanced uptake of a thermally responsive polypeptide by tumor cells in response to its hyperthermia-mediated phase transition. Cancer Res. 61, 7163–7170. (19) Shiotani, A., Mori, T., Niidome, T., Niidome, Y., and Katayama, Y. (2007) Stable incorporation of gold nanorods into N-isopropylacrylamide hydrogels and their rapid shrinkage induced by near-IR laser irradiation. Langmuir 23, 4012–4018. (20) Gorelikov, I., Field, L. M., and Kumacheva, E. (2004) Hybrid microgels photoresponsive in the near-infrared spectral range. J. Am. Chem. Soc. 126, 15938–15939. (21) Das, M., Mordoukhovski, L., and Kumacheva, E. (2008) Sequestering gold nanorods by polymer microgels. AdV. Mater. 20, 2371–2375. (22) Niidome, Y., Nishioka, K., Kawasaki, H., and Yamada, S. (2003) Rapid synthesis of gold nanorods by the combination of chemical reduction and photoirradiation processes; morphological changes depending on the growing processes. Chem. Commun. 18, 2376–2377. (23) Nikoobakht, B., and El-sayed, M. A. (2001) Evidence for bilayer assembly of cationic surfactants on the surface of gold nanorods. Langmuir 17, 6368–6374. (24) Gao, J., Bender, C. M., and Murphy, C. J. (2003) Dependence of the gold nanorod aspect ratio on the nature of the directing surfactant in aqueous solution. Langmuir 19, 9065–9070.
Communications (25) Niidome, T., Yamagata, M., Okamoto, Y., Akiyama, Y., Takahashi, H., Kawano, T., Katayama, Y., and Niidome, Y. (2006) PEG-modified gold nanorods with a stealth character for in ViVo applications. J. Controlled Release 114, 343–347. (26) Pastoriza-Santos, I., Pe´rez-Juste, J., and Liz-Marza´n, L. M. (2006) Silica-coating and hydrophobation of CTAB-stabilized gold nanorods. Chem. Mater. 18, 2465–2467. (27) Sto¨ber, W., Fink, A., and Bohn, E. J. (1968) Controlled growth of monodisperse silica spheres in the micron size. Range. Colloid Interface Sci. 26, 62–69. (28) Link, S., and El-Sayed, M. A. (1999) Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods. J. Phys. Chem. B 103, 8410–8426. (29) Liz-Marza´n, L. M., Giersig, M., and Mulvaney, P. (1996) Synthesis of nanosized gold-silica core-shell particles. Langmuir 12, 4329–4335. (30) Deng, Y., Yang, W., Wang, C., and Fu, S. (2003) A novel approach for preparation of thermoresponsive polymer magnetic microspheres with core-shell structure. AdV. Mater. 15, 1729– 1732. (31) Singh, N., and Lyon, L. A. (2007) Au nanoparticle templated synthesis of pNIPAm nanogels. Chem. Mater. 19, 719–726. (32) Wang, H., Huff, T. B., Zweifel, D. A., He, W., Low, P. S., Wei, A., and Cheng, J. X. (2005) In vitro and in vivo two-photon luminescence imaging of single gold nanorods. Proc. Natl. Acad. Sci. U.S.A. 102, 15752–15756. (33) Niidome, T., Akiyama, Y., Shimoda, K., Kawano, T., Mori, T., Katayama, Y., and Niidome, Y. (2008) In vivo monitoring of intravenous injected gold nanorods using near-infrared light. Small 4, 1001–1007. BC800480K
