Rapid communicationPEG-modified gold nanorods with a stealth character for in vivo applications
Introduction
Gold nanoparticles show unique optical properties such as distinctive extinction bands in the visible region, due to surface plasmon (SP) oscillation of free electrons [1]. This property allows the use of nanoparticles for many applications, e.g., they can be used as Raman sensors [2], photocatalysts [3], and photoelectrochemical materials [4], [5]. In the bioscience and medical fields, gold nanoparticles are used as immunostaining marker particles for electron microscopy, and as chromophores for immunoreactions and nucleic acid hybridization [6], [7]. Recently, their application for gene delivery into cells was reported [8], [9], [10], [11]. In addition, gold nanoparticles have attracted much attention as photothermal agents in hyperthermia [12]. Hyperthermia causes cellular necrosis from an increase in temperature at local areas by irradiating laser light [13], [14], microwaves [15], radiofrequency [16], and focused ultrasound [17]. Since gold nanoparticles show strong absorbance in the visible to near-infrared region, it is expected that a therapeutic dose of heat could be delivered to local areas, where nanoparticles exist, by extracorporeally irradiating with a laser light. El-Sayed et al. modified gold nanoparticles with an anti-EGFR antibody, and achieved tumor imaging using light scattering from nanoparticles in cells, and specific cell damage combined with irradiation of continuous waves (CW) of an argon laser [12], [18]. Their in vitro demonstrations of imaging and therapy of tumor cells provided insights for application of gold nanoparticles to the clinics.
The use of gold-coated silica nanoshells as imaging and therapeutic agents has preceded [19], [20], [21] that of gold nanoparticles. Optical resonances of nanoshells can be continuously tuned for wavelengths ranging between the ultraviolet and infrared regions [22]. In particular, the near infrared region (650–900 nm) is ideally suited for in vivo imaging and therapy due to minimum light absorption by intrinsic chromophores, hemoglobin (< 650 nm), and water (> 900 nm), indicating maximal penetration of light into tissues [23]. Using nanoshells tuned to an absorbance at 820 nm, Hirsch et al. achieved photothermal ablation of tumor cells by irradiation using moderate near infrared laser light in vivo [20].
Gold nanorods, rod-shaped gold nanoparticles, have unique optical properties [24], [25]. They show two surface plasmon bands corresponding to the transverse and longitudinal surface plasmon bands in the visible (∼ 520 nm) and the near-infrared regions, respectively. The longitudinal band has a substantially larger extinction coefficient than the transverse band. Thus, gold nanorods are unusual materials with an intense surface plasmon band that affords absorption, fluorescence [26], [27], and light scattering [28], [29] in the near infrared region, and near-IR, inducing two-photon luminescence [30].
Despite their unique characteristics, gold nanorods cannot be used in the bioscience field due to strong cytotoxic hexadecyltrimethylammonium bromide (CTAB), a cationic detergent used as the stabilizing agent during preparation of gold nanorods [24]. To reduce CTAB cytotoxicity, gold nanoparticles in solution are washed by centrifugation [31]. However, CTAB bilayers usually remain on the surface of gold nanorods, and CTAB bilayers non-covalently adsorb onto the surface, indicating that further removal of CTAB will result in aggregation of nanorods. To reduce cytotoxicity of gold nanorods, and stabilize them in biocompatible conditions, we developed a technique to remove CTAB, and modified nanorods with phosphatidylcholine (PC), i.e., CTAB was extracted from the nanorod solution into the chloroform phase containing phosphatidylcholine [32]. Recently, Huang et al. demonstrated tumor imaging and photothermal therapy by combining infrared light and gold nanorods as light scattering and photothermal agents, respectively in an in vitro system [33]. Anti-EGFR antibody-modified gold nanorods specifically bound to malignant cell lines expressing EGFR, showing strong light scattering from adsorbed nanorods on cells. Irradiation with a CW laser at 800 nm caused damage of cells bound with nanorods, indicating that gold nanorods were candidate functional agents for dual imaging and therapy of tumor cells. On the other hand, gold nanorods have been used as agents for controlled release of DNA into cells [34]. Thiol-modified EGFP-encoded DNA fragments amplified by PCR were modified onto nanorods. After irradiation with femto-second near infrared pulse laser light, DNA was released from nanorods due to transformation of nanorods to spherical particles, then, EGFP expression was observed without significant cytotoxicity.
To apply gold nanorods to medical fields including tumor imaging, photothermal therapy, gene delivery, and drug delivery, targeted delivery of nanorods after systemic injection is a key parameter. For the targeted delivery in vivo, a stealth character is certainly required. Providing the stealth character to nanorods will enable efficient delivery to a specific site. It will also produce higher contrast images, and a more effective photothermal therapy compared to current techniques.
Gold nanorods are advantageous due to their strong and sharp surface plasmon bands in the near-infrared region, and these peak positions are tunable depending on their shapes [24]. Easy preparation and modification of the surface with many functional groups such as a ligand for targeted delivery, are also advantageous characters of gold nanorods compared to nanoshells. In this study, we prepared gold nanorods modified with polyethyleneglycol (PEG), then, evaluated its cytotoxicity in vitro and biodistribution after intravenous injection into mice. We also discussed possible medical applications of nanorods such as tumor imaging and photothermal therapy.
Section snippets
Preparation of surface-modified gold nanorods
Gold nanorods, prepared with a modification of our previous methods [35], were supplied by Mitsubishi Materials Co. Ltd. Average length and width of as-prepared gold nanorods were 65 ± 5 nm and 11 ± 1 nm, respectively (aspect ratio: 5.9) [32]. PEG-modified gold nanorods were prepared as follows. A solution of gold nanorods containing CTAB was centrifuged at 14,000 g for 10 min, decanted, and resuspended in water to remove excess CTAB. 200 μl 5-mM mPEG5000-SH were added to 1 ml 1-mM gold nanorod
Surface modification of gold nanorods
PEG-modified gold nanorods were prepared by mixing mPEG-SH with gold nanorods stabilized by CTAB. After reacting the thiol group of PEG with the surface of gold nanorods, CTAB was removed by dialysis against water. Absorption spectra of PEG-modified gold nanorod solution were measured (Fig. 1). The spectra showed typical characteristics of a NR solution, i.e., two surface plasmon bands corresponding to the longitudinal (900 nm) and transverse (520 nm) oscillation modes were seen [32].
Discussion
Targeted delivery of gold nanorods following systemic injection is an important technique for future medical applications, e.g., in vivo imaging of target sites using near infrared light scattering and photothermal therapy using near infrared laser light. In this study, we attempted to develop gold nanorods with a stealth character in vivo using mice as a model, in which gold nanorods were systemically injected into the tail vein. We compared two kinds of gold nanorods (stabilized by CTAB and
Acknowledgements
This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 16790106), and a Grant-in-Aid for Scientific Research in the Priority Area “Molecular Nano Dynamics” (No. 17034049) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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